Highly Sensitive Biomarker Panels

ABSTRACT

Cardiovascular disease, e.g., congestive heart failure, is often first diagnosed after the onset of clinical symptoms, eliminating potential for early intervention. The invention provides a multi-marker immunoassay, including cardiac pathology and vascular inflammation biomarkers, yielding a more sensitive assay for early detection of CHF in plasma. A panel consisting of cardiac pathology (cTnI, BNP) and vascular inflammation (IL-6, TNFα, IL-17a) biomarkers provided a sensitivity of 94% for association with CHF.

BACKGROUND OF THE INVENTION

Cardiovascular disease is an abnormal function of the heart and/or bloodvessels. Included under this designation are such diverse medicalconditions as coronary artery disease, congestive heart failure,arrhythmia, atherosclerosis, hypertension, stroke, cerebrovasculardisease, peripheral vascular disease and myocardial infarction. In theUnited States, CVD is a major cause of death. About 40 percent of alldeaths in 1997, or about one million people, were attributed tocardiovascular disease. There are an estimated 62 million people withcardiovascular disease and 50 million people with hypertension in thiscountry.

Cardiovascular disease is a progressive process with etiologies in bothcardiac muscle (cardio-pathology) and vascular inflammation. The diseaseprocess follows a continuum from early onset mild vascular inflammationto severe acute events such as acute myocardial infarction or chronicevents such as heart failure. Patients with well recognized physicalconditions such as hypertension, obesity, diabetes, metabolic syndrome,hyper-cholesterolemia are at varying degrees of risk for developing CVD.A challenge facing clinicians who have patients presenting with CVD riskfactors is understanding their degree of risk, developing theappropriate treatment plan and then monitoring the patient forimprovements in disease risk.

Ample studies have provided compelling evidence that CVD is largelypreventable. The causes of cardiovascular disease range from structuraldefects, to infection, inflammation, environment and genetics. Whilesome risk factors cannot be modified (genetics, age, gender), there area number of risk factors that can be addressed through lifestyle changesor medically. These controllable risk factors include cigarette smoking,high blood pressure, obesity, diabetes, physical inactivity, and highblood cholesterol level. By the time that heart problems are detected,the underlying cause (atherosclerosis) is usually quite advanced, havingprogressed for decades. There is therefore increased emphasis onpreventing atherosclerosis by modifying risk factors, such as healthyeating, exercise and avoidance of smoking.

CVD, e.g., congestive heart failure (CHF), is often first diagnosedafter the onset of clinical symptoms, eliminating potential for earlyintervention. There is a need for highly sensitive detection of CVD.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method for detectingor monitoring a condition in a subject, comprising detecting a firstmarker in a first sample from the subject and detecting a second marker,wherein the first marker comprises Cardiac Troponin-I (cTnI) or VascularEndothelial Growth Factor (VEGF), and wherein the limit of detection ofthe first marker is less than about 20 pg/ml. In some embodiments, thedetection of at least one marker comprises contacting the sample with alabel for the marker and detecting the presence or absence of the label,wherein detection of the presence of the label indicates the presence ofthe corresponding marker. In some embodiments, the label comprises afluorescent moiety, and the detection comprises passing the labelthrough a single molecule detector, wherein the single molecule detectorcomprises: (a) an electromagnetic radiation source for stimulating thefluorescent moiety; (b) an interrogation space for receivingelectromagnetic radiation emitted from the electromagnetic source; and(c) an electromagnetic radiation detector operably connected to theinterrogation space for determining an electromagnetic characteristic ofthe stimulated fluorescent moiety.

In some embodiments, the limit of detection of the first marker rangesfrom about 10 pg/ml to about 0.01 pg/ml. In some embodiments, the limitof detection of the first marker is less than about 10 pg/ml. In someembodiments, the limit of detection of the first marker is less thanabout 5 pg/ml. In some embodiments, the limit of detection of the firstmarker is less than about 1 pg/ml. In some embodiments, the limit ofdetection of the first marker is less than about 0.5 pg/ml. In someembodiments, the limit of detection of the first marker is less thanabout 0.1 pg/ml. In some embodiments, the limit of detection of thefirst marker is less than about 0.05 pg/ml. In some embodiments, thelimit of detection of the first marker is less than about 0.01 pg/ml. Insome embodiments, the limit of detection of the first marker is lessthan about 0.005 pg/ml. In some embodiments, the limit of detection ofthe first marker is less than about 0.001 pg/ml. In some embodiments,the coefficient of variation (CV) of the limit of detection ranges fromabout 20% to about 1%. In some embodiments, the coefficient of variation(CV) of the limit of detection ranges from about 100% to about 1%. Insome embodiments, the coefficient of variation (CV) of the limit ofdetection ranges from about 75% to about 1%. In some embodiments, thecoefficient of variation (CV) of the limit of detection ranges fromabout 50% to about 1%. In some embodiments, the coefficient of variation(CV) of the limit of detection ranges from about 25% to about 1%. Insome embodiments, the coefficient of variation (CV) of the limit ofdetection ranges from about 20% to about 1%. In some embodiments, thecoefficient of variation (CV) of the limit of detection ranges fromabout 15% to about 1%. In some embodiments, the coefficient of variation(CV) of the limit of detection ranges from about 10% to about 1%. Insome embodiments, the coefficient of variation (CV) of the limit ofdetection ranges from about 5% to about 1%. In some embodiments, thesample size ranges from about 10 μl to about 0.1 μl. In someembodiments, the sample size ranges from about 100 μl to about 0.1 μl.In some embodiments, the sample size ranges from about 75 μl to about0.1 μl. In some embodiments, the sample size ranges from about 50 μl toabout 0.1 μl. In some embodiments, the sample size ranges from about 25μl to about 0.1 μl. In some embodiments, the sample size ranges fromabout 20 μl to about 0.1 μl. In some embodiments, the sample size rangesfrom about 5 μl to about 0.1 μl. In some embodiments, the sample sizeranges from about 1 μl to about 0.1 μl. In some embodiments, the samplesize is less than about 100 μl. In some embodiments, the sample size isless than about 75 μl. In some embodiments, the sample size is less thanabout 50 μl. In some embodiments, the sample size is less than about 25μl. In some embodiments, the sample size is less than about 20 μl. Insome embodiments, the sample size is less than about 15 μl. In someembodiments, the sample size is less than about 10 μl. In someembodiments, the sample size is less than about 5 μl. In someembodiments, the sample size is less than about 2 μl. In someembodiments, the sample size is less than about 1 μl. In someembodiments, the sample size is less than about 0.5 μl. In someembodiments, the sample size is less than about 0.1 μl. In someembodiments, the sample size is less than about 0.05 μl. In someembodiments, the sample size is less than about 0.01 μl.

In some embodiments, the method further comprises splitting the firstsample into two or more aliquots and detecting at least one marker inthe two or more aliquots. In some embodiments, the sample comprises aplasma, serum, cell lysate, or tissue sample. In some embodiments, thesample comprises bronchoalveolar lavage fluid (BAL), blood, serum,plasma, urine, nasal swab, cerebrospinal fluid, pleural fluid, synovialfluid, peritoneal fluid, amniotic fluid, gastric fluid, lymph fluid,interstitial fluid, tissue homogenate, cell extracts, saliva, sputum,stool, physiological secretions, tears, mucus, sweat, milk, semen,seminal fluid, vaginal secretions, fluid from ulcers and other surfaceeruptions, blisters, and abscesses, and extracts of tissues includingbiopsies of normal, malignant, and suspect tissues or any otherconstituents of the body which may contain the target particle ofinterest. Other similar specimens such as cell or tissue culture orculture broth are also of interest.

In some embodiments, the second marker comprises a biomarker, aphysiological marker or a genetic marker. In some embodiments, thesecond marker comprises a protein. In some embodiments, at least one ofthe first marker and the second marker are found in a sample from anormal individual at a concentration of less than 10 pg/ml. In someembodiments, at least one of the first marker and the second marker arefound in a sample from a normal individual at a concentration of lessthan 100 pg/ml. In some embodiments, at least one of the first markerand the second marker are found in a sample from a normal individual ata concentration of less than 75 pg/ml. In some embodiments, at least oneof the first marker and the second marker are found in a sample from anormal individual at a concentration of less than 50 pg/ml. In someembodiments, at least one of the first marker and the second marker arefound in a sample from a normal individual at a concentration of lessthan 25 pg/ml. In some embodiments, at least one of the first marker andthe second marker are found in a sample from a normal individual at aconcentration of less than 20 pg/ml. In some embodiments, at least oneof the first marker and the second marker are found in a sample from anormal individual at a concentration of less than 15 pg/ml. In someembodiments, at least one of the first marker and the second marker arefound in a sample from a normal individual at a concentration of lessthan 10 pg/ml. In some embodiments, at least one of the first marker andthe second marker are found in a sample from a normal individual at aconcentration of less than 5 pg/ml. In some embodiments, at least one ofthe first marker and the second marker are found in a sample from anormal individual at a concentration of less than 2 pg/ml. In someembodiments, at least one of the first marker and the second marker arefound in a sample from a normal individual at a concentration of lessthan 1 pg/ml. In some embodiments, at least one of the first marker andthe second marker are found in a sample from a normal individual at aconcentration of less than 0.5 pg/ml. In some embodiments, at least oneof the first marker and the second marker are found in a sample from anormal individual at a concentration of less than 0.1 pg/ml. In someembodiments, at least one of the first marker and the second marker arefound in a sample from a normal individual at a concentration of lessthan 0.05 pg/ml. In some embodiments, at least one of the first markerand the second marker are found in a sample from a normal individual ata concentration of less than 0.01 pg/ml.

In some embodiments, the limit of detection of the second marker rangesfrom about 10 pg/ml to about 0.01 pg/ml. In some embodiments, the limitof detection of the second marker is less than about 10 pg/ml. In someembodiments, the limit of detection of the second marker is less thanabout 5 pg/ml. In some embodiments, the limit of detection of the secondmarker is less than about 1 pg/ml. In some embodiments, the limit ofdetection of the second marker is less than about 0.5 pg/ml. In someembodiments, the limit of detection of the second marker is less thanabout 0.1 pg/ml. In some embodiments, the limit of detection of thesecond marker is less than about 0.05 pg/ml. In some embodiments, thelimit of detection of the second marker is less than about 0.01 pg/ml.In some embodiments, the limit of detection of the second marker is lessthan about 0.005 pg/ml. In some embodiments, the limit of detection ofthe second marker is less than about 0.001 pg/ml.

In some embodiments, the second marker comprises B-type natiureticpeptide, IL-1α, IL-1β, IL-6, IL-8, IL-10, TNF-α, IFN-γ, cTnI, VEGF,insulin, GLP-1 (active), GLP-1 (total), TREM1, Leukotriene E4, Akt1,Aβ-40, Aβ-42, Fas ligand, or PSA. In some embodiments, the second markeris a cytokine. In some embodiments, the cytokine is G-CSF, MIP-1α,IL-10, IL-22, IL-8, IL-5, IL-21, INF-γ, IL-15, IL-6, TNF-α, IL-7,GM-CSF, IL-2, IL-4, IL-1α, IL-12, IL-17α, IL-1β, MCP, IL-32 or RANTES.In some embodiments, the cytokine is IL-10, IL-8, INF-γ, IL-6, TNF-α,IL-7, IL-1α, or IL-1β. In some embodiments, the second marker comprisesan apolipoprotein, ischemia-modified albumin (IMA), fibronectin,C-reactive protein (CRP), B-type Natriuretic Peptide (BNP), orMyeloperoxidase (MPO).

In some embodiments, the method of the invention further comprisesdetermining a concentration for the first marker, and determining aconcentration for the second marker if the second marker comprises aprotein. In some embodiments, the method of the invention comprisesdetermining a ratio of a concentration of the first marker compared to aconcentration for the second marker if the second marker comprises aprotein.

In some embodiments, the second marker comprises a physiological marker.In some embodiments, the physiological marker comprises anelectrocardiogram (EKG), stress testing, nuclear imaging, ultrasound,insulin tolerance, body mass index, blood pressure, age, sex, or sleepapnea.

In some embodiments, the second marker comprises a molecular marker. Insome embodiments, the molecular marker comprises cholesterol,LDL/HDL/Q-LDL, triglycerides, uric acid, creatinine, blood glucose orvitamin-D. In some embodiments, the molecular marker comprisessubfractions of LDL/HDL/Q-LDL or triglycerides.

In some embodiments, the second marker comprises a genetic marker. Insome embodiments, the genetic marker comprises a variation in a geneencoding an apolipoprotein such as ApoE. In some embodiments, thegenetic marker comprises a single nucleotide polymorphism (SNP). In someembodiments, the genetic marker comprises an insertion, deletion, fusionor other mutation. In some embodiments, the genetic marker comprises anepigenetic marker, such as DNA methylation or imprinting.

In some embodiments of the method of the invention, the conditioncomprises cardiac damage, an inflammatory disease, a proliferativedisorder, a metabolic disorder, angiogenesis, artherosclerosis ordiabetes. In some embodiments, the cardiac damage comprises myocardialinfarct, necrosis, myocardial dysfunction, unstable angina, plaques,heart failure, coronary artery disease, or rheumatic heart disease. Insome embodiments, the proliferative disorder comprises a cancer. In someembodiments, the cancer comprises a breast cancer, a prostate cancer, orlymphoma.

In some embodiments, the method of the invention further comprisesdetermining a change in concentration of the markers between the firstsample and a second sample from the subject, whereby the change is usedto detect or monitor the condition. In some embodiments, the method ofthe invention further comprises determining a change in the ratio of theconcentrations of the first marker and the second marker between thefirst sample and a second sample from the subject, whereby the change isused to detect or monitor the condition. In some embodiments, a medicalprocedure is performed between acquiring the first sample and the secondsample from the subject. In some embodiments, the medical procedurecomprises a surgical procedure, stress testing or a therapeuticintervention. In some embodiments, a series of samples from the subjectare used to detect or monitor the condition. In some embodiments, theseries of samples are collected over time and the change ofconcentration in the series of samples is assessed.

In some embodiments, monitoring according to the present inventioncomprises monitoring of a disease progression, disease recurrence, riskassessment, therapeutic efficacy or surgical efficacy.

In one embodiment, the present invention provides a method for detectinga single particle in a sample, comprising: (a) labeling the particle, ifpresent in the sample, with a label; and (b) detecting the presence orabsence of the label, wherein detection of the presence of the labelindicates the presence of the single particle in the sample; wherein thelimit of detection of the single particle is less than 20 pg/ml; andwherein the single particle comprises a single molecule, fragment, orcomplex of Cardiac Troponin-I (cTnI), B-type Natriuretic Peptide (BNP,proBNP or NT-proBNP), TREM-1, Interleukin 1 Alpha (IL-1α), Interleukin 1Beta (IL-1β), Interleukin 4 (IL-4), Interleukin 6 (IL-6), Interleukin 8(IL-8), Interleukin 10 (IL-10), Interferon gamma (IFN-γ), Tumor NecrosisFactor alpha (TNF-α), Glucagon-like peptide-1 (GLP-1), Leukotriene E4(LTE4), Transforming Growth Factor Beta (TGFβ), Akt1, Aβ-40, Aβ-42, Fasligand (FasL), or Vascular Endothelial Growth Factor (VEGF). In someembodiments, the limit of detection of the single particle rangesbetween about 10 pg/ml and about 0.01 pg/ml. In some embodiments, thelimit of detection of the single particle is less than about 10 pg/ml.In some embodiments, the limit of detection of the single particle isless than about 5 pg/ml. In some embodiments, the limit of detection ofthe single particle is less than about 1 pg/ml. In some embodiments, thelimit of detection of the single particle is less than about 0.5 pg/ml.In some embodiments, the limit of detection of the single particle isless than about 0.1 pg/ml. In some embodiments, the limit of detectionof the single particle is less than about 0.05 pg/ml. In someembodiments, the limit of detection of the single particle is less thanabout 0.01 pg/ml. In some embodiments, the limit of detection of thesingle particle is less than about 0.005 pg/ml. In some embodiments, thelimit of detection of the single particle is less than about 0.001pg/ml.

In one embodiment, the present invention provides a kit comprising acomposition comprising two or more antibodies to two or more biomarkers,wherein the two or more antibodies are attached to a fluorescent dyemoiety, wherein the two or more biomarkers comprise particles asdescribed above, wherein the moiety is capable of emitting at leastabout 200 photons when stimulated by a laser emitting light at theexcitation wavelength of the moiety, wherein the laser is focused on aspot not less than about 5 microns in diameter that contains the moiety,and wherein the total energy directed at the spot by the laser is nomore than about 3 microJoules, wherein the composition is packaged insuitable packaging.

In one embodiment, the present invention provides a method for detectingor monitoring a cardiovascular condition in a subject, comprisingdetecting two or more biomarkers, wherein the biomarkers are cardiacpathology or vascular inflammation biomarkers, and wherein the limit ofdetection of at least one marker is less than about 20 pg/ml. In someembodiments, the detection of at least one marker comprises contactingthe sample with a label for the marker and detecting the presence orabsence of the label, wherein detection of the presence of the labelindicates the presence of the corresponding marker. In some embodiments,the label comprises a fluorescent moiety, and the detection comprisespassing the label through a single molecule detector, wherein the singlemolecule detector comprises: a) an electromagnetic radiation source forstimulating the fluorescent moiety; b) an interrogation space forreceiving electromagnetic radiation emitted from the electromagneticsource; and c) an electromagnetic radiation detector operably connectedto the interrogation space for determining an electromagneticcharacteristic of the stimulated fluorescent moiety. In someembodiments, the limit of detection of at least one marker ranges fromabout 10 pg/ml to about 0.01 pg/ml.

In some embodiments, the cardiac pathology biomarkers are CardiacTroponin-I (cTnI) or B-type Natriuretic Peptide (BNP, proBNP orNT-proBNP). In some embodiments, the vascular inflammation biomarkersare Interleukin 6 (IL-6), Tumor Necrosis Factor alpha (TNF-α), orInterleukin 17a (IL-17a). In some embodiments, the condition comprisescongestive heart failure and the biomarkers comprise cTnI, BNP, IL-6,TNF-α, and IL-17a.

In one embodiment, the present invention provides a method to detect acardiovascular condition in a subject, comprising measuring aphysiological biomarker and detecting one or more biomarkers in a bloodsample from the subject. In some embodiments, the physiologicalbiomarker is a stress test and the one or more biomarkers comprise cTnI.In some embodiments, the physiological biomarker is a sleep test and theone or more biomarkers comprise one or more cytokines. The one or morecytokines can comprise TNF-a, IL-6, IL-17a and/or hsCRP. In someembodiments, the physiological biomarker is Carotid intima-mediathickness (CIMT) and the one or more biomarkers comprise one or moremarkers of vascular inflammation. The one or more markers of vascularinflammation can comprise hsCRP, IL-6, TNF-a and/or IL-1.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A and 1B illustrate schematic diagrams of the arrangement of thecomponents of a single particle analyzer. FIG. 1A shows an analyzer thatincludes one electromagnetic source and one electromagnetic detector;FIG. 1B shows an analyzer that includes two electromagnetic sources andone electromagnetic detector.

FIGS. 2A and 2B illustrate schematic diagrams of a capillary flow cellfor a single particle analyzer. FIG. 2A shows the flow cell of ananalyzer that includes one electromagnetic source; and FIG. 2B shows theflow cell of an analyzer that includes two electromagnetic sources.

FIGS. 3A and 3B illustrate schematic diagrams showing the conventional(A) and confocal (B) positioning of laser and detector optics of asingle particle analyzer. FIG. 3A shows the arrangement for an analyzerthat has one electromagnetic source and one electromagnetic detector;FIG. 3B shows the arrangement for an analyzer that has twoelectromagnetic sources and two electromagnetic detectors.

FIG. 4 illustrates a flow chart for multiple marker detection ormonitoring of a condition.

FIG. 5 illustrates a computer system wherein a client workstationreceives assay results from a remote computer.

FIG. 6 illustrates a linearized standard curve for the rangeconcentrations of cTnI.

FIG. 7A is a graph illustrating the analytical sensitivity of cTnI of a100 μl sample and a 50 μl sample at an LoD of 0.1-0.2 pg/ml. FIG. 7B isa graph illustrating the low end of a standard curve signal.

FIG. 8 illustrates a biological threshold (cutoff concentration) forcTnI at a cTnI concentration of 7 pg/ml, as established at the 99thpercentile with a corresponding coefficient of variation (CV) of 10%.

FIG. 9 illustrates a correlation of assay results of cTnI determinedusing the analyzer system of the invention with standard measurementsprovided by the National Institute of Standards and Technology (NIST)(R²=0.9999).

FIG. 10 illustrates detection of cTnI in serial serum samples frompatients who presented at the emergency room with chest pain. Themeasurements made with the analyzer system of the invention werecompared to measurements made with a commercially available assay.

FIG. 11 illustrates distribution of normal biological concentrations ofcTnI and concentrations of cTnI in serum samples from patientspresenting with chest pain.

FIG. 12 illustrates a competition curve for LTE4. The LOD was determinedto be 1.5 pg/ml LTE4.

FIG. 13 illustrates a graph showing the standard curve forconcentrations of Akt1. The LOD was calculated to be 25 pg/ml Akt1.

FIG. 14 illustrates a graph showing the standard curve forconcentrations of TGFβ. The LOD was calculated to be 350 pg/ml TGFβ.

FIG. 15 illustrates a schematic representation of a kit that includes ananalyzer system for detecting a single protein molecule in a sample andleast one label that includes a fluorescent moiety and a binding partnerfor the protein molecule, where the analyzer includes an electromagneticradiation source for stimulating the fluorescent moiety; a capillaryflow cell for passing the label; a source of motive force for moving thelabel in the capillary flow cell; an interrogation space defined withinthe capillary flow cell for receiving electromagnetic radiation emittedfrom the electromagnetic source; and an electromagnetic radiationdetector operably connected to the interrogation space for measuring anelectromagnetic characteristic of the stimulated fluorescent moiety,where the fluorescent moiety is capable of emitting at least about 200photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot not lessthan about 5 microns in diameter that contains the moiety, and where thetotal energy directed at the spot by the laser is no more than about 3microJoules.

FIG. 16 illustrates a standard curve of TREM-1 measured in a sandwichmolecule immunoassay developed for the single particle analyzer system.The linear range of the assay is 100-1500 fM.

FIGS. 17A-F illustrate detection of IL-6 and IL-8. A) IL-6 standards,diluted according to a commercially available kit (R&D Systems,Minneapolis, Minn.) gave a linear response between 0.1 and 10 pg/ml. B)IL-6 standard curve below 1 pg/ml. C) and D) Distribution of IL-6 C) andIL-8 D) identified in blood bank donor EDTA specimens. E) Range ofdetection at low concentrations of any analyte can be extended to higherconcentrations by switching the detection of the analyzer from countingmolecules (digital signal) to detecting the sum of photons (analogsignal) that are generated at the higher concentrations of analyte. Thesingle particle analyzer has an expanded linear dynamic range of 6 logs.Six-log range of detection based on switching from digital to analogdetection. F) Non-linearized standard curve showing range of lowconcentrations of IL-6 (0.1 fg/ml-10 fg/ml) determined by countingphotons emitted by individual particles (digital signal), and higherrange of concentrations of IL-6 (10 fg/ml-1 pg/ml).

FIG. 18 illustrates a comparison of assays of the invention withconventional assays.

FIG. 19A is a graph illustrating the performance of a human VEGF assay;FIG. 19B is a graph of the assay performance at the lowestconcentrations.

FIG. 20A is a graph illustrating the performance of a mouse VEGF assay;FIG. 20B is a graph of the assay performance at the lowestconcentrations.

FIG. 21 is a graph comparing the VEGF assays of the present inventionand ELISA assays of human plasma.

FIG. 22A is a graph comparing the level of VEGF detected in cell lysatesand culture media using MDA-MB-231 breast adenocarcinoma cells; FIG. 22Bis a graph comparing the level of VEGF detected in cell lysates andculture media using HT-29 colon adenocarcinoma cells.

FIG. 23 is a comparison of VEGF assays of the present invention andELISA assays for mouse plasma samples.

FIG. 24A is a graph illustrating the concentration of mouse VEGFdetected in cell lysates and culture media using B16 melanoma mouse celllines; FIG. 24B is a graph illustrating the concentration of mouse VEGFdetected in cell lysates and culture media using 4T1 mammary carcinoma;FIG. 24C is a graph illustrating the concentration of mouse VEGFdetected in cell lysates and culture media using CT26 colon carcinomacell lines.

FIG. 25A illustrates a graph showing highly sensitive detection of VEGF.FIG. 25B illustrates the low end standard curve signal.

FIG. 26 illustrates the measured versus expected levels of detection ofhuman VEGF using three different immunoassay formats: 1) MagneticMicroparticle based Single Molecule Counting (MP-SMC); 2) 384-well Platebased Single Molecule Counting (Plate-SMC); and 3) Horseradishperoxidase based Enzyme Linked Immunosorbent Assay (HRP-ELISA).

FIG. 27A illustrates the levels of human VEGF detected in 10 μl plasmasamples from healthy and breast cancer patients. The limit of detection(LOD) using the method of the present invention (Errena; LOD=3.5 pg/ml)versus a standard ELISA format (LOD=31.2 pg/ml) is shown. FIG. 27Billustrates similar data in 10 μl lysate samples.

FIG. 28A-C illustrates combined analog and digital measurements of VEGF.

FIG. 29A illustrates a graph showing the specificity and linearity ofAβ-40 assay. FIG. 29B is a graph showing the specificity and linearityof an Aβ-42 assay.

FIG. 30A is a graph illustrating an assay curve fit for IL-1α. FIG. 30Bis a graph illustrating the low end of an IL-1αassay standard curvesignal.

FIG. 31A is a graph illustrating an IL-1β assay curve fit. FIG. 31Billustrates the low end standard curve of IL-1β curve signal.

FIG. 32A is a graph illustrating an IL-4 assay curve fit. FIG. 32B is anIL-4 assay standard curve signal at the low end.

FIG. 33A is a graph illustrating an IL-6 assay curve fit. FIG. 33B is anIL-6 assay standard curve signal at the low end.

FIG. 34 illustrates diagnostic specificity of the cardiovascular disease(CVD) panel for conjestive heart failure (CHF) subjects compared to ageand sex matched healthy control subjects.

FIG. 35 illustrates the number of elevated biomarkers for thecardiovascular disease (CVD) panel in conjestive heart failure (CHF)subjects.

DETAILED DESCRIPTION OF THE INVENTION Outline I. Introduction II.Molecules for Sensitive Detection By the Methods and Compositions of theInvention

A. General

B. Markers

III. Labels

A. Binding partners

1. Antibodies

B. Fluorescent Moieties

1. Dyes

2. Quantum dots

C. Binding Partner-Fluorescent Moiety Compositions

IV. Highly Sensitive Analysis of Molecules

A. Sample

B. Sample preparation

C. Detection of molecule of interest and determination of concentration

V. Instruments and Systems Suitable for Highly Sensitive Analysis ofMolecules

A. Apparatus/System

B. Single Particle Analyzer

1. Electromagnetic Radiation Source

2. Capillary Flow Cell

3. Motive Force

4. Detectors

C. Sampling System

D. Sample preparation system

E. Sample recovery

VI. Methods Using Highly Sensitive Analysis of Molecules

A. Methods

B. Exemplary Markers

1. Cardiac damage

2. Infection

3. Cytokines

a. Interleukin 1

b. Interleukin 4

c. Interleukin 6

4. Inflammatory Markers

a. Leukotrine E4

b. TGFβ

5. Akt1

6. Fas ligand

7. VEGF

8. Amyloid beta proteins

C. Multiple Marker Panels

1. Multiple Biomarker Panels

2. Mixed Marker Panels

D. Detection and Monitoring

E. Cardiovascular Biomarker Panels

F. Clinical Methods

VII. Kits VIII. Examples I. INTRODUCTION

The invention provides instruments, kits, compositions, and methods forthe highly sensitive detection of single molecules, and for thedetermination of the concentration of the molecules in a sample. In someembodiments, the sensitivity and precision of the instruments,compositions, methods, and kits of the invention can be achieved by acombination of factors selected from, but not limited to,electromagnetic sources of appropriate wavelength and power output,appropriate interrogation space size, high numerical aperture lenses,detectors capable of detecting single photons, and data analysis systemsfor counting single molecules. The instruments of the invention arereferred to as “single molecule detectors” or “single particledetectors,” and are also encompassed by the terms “single moleculeanalyzers” and “single particle analyzers.” The sensitivity andprecision of the kits and methods of the invention are achieved in someembodiments by the use of the instruments of the invention together witha combination of factors selected from, but not limited to, labels formolecules that exhibit characteristics that allow the molecules to bedetected at the level of the single molecule, and methods assaying thelabel in the instruments described herein.

The instruments, kits, and methods of the invention are especiallyuseful in the sensitive and precise detection of single molecules orsmall molecules, and for the determination of the concentration of themolecules in a sample.

The invention provides, in some embodiments, instruments and kits forthe sensitive detection and determination of concentration of moleculesby detection of single molecules, labels for such detection anddetermination, and methods using such instruments and labels in theanalysis of samples. In particular, the sensitivity and precision of theinstruments, kits, and methods of the invention make possible thedetection and determination of concentration of molecules, e.g., markersfor biological states, at extremely low concentrations, e.g.,concentrations below about 100, 10, 1, 0.1, 0.01, or 0.001 femtomolar.In further embodiments, the instruments and kits of the invention arecapable of determining a concentration of a species in a sample, e.g.,the concentration of a molecule, over a large dynamic range ofconcentrations without the need for dilution or other treatment ofsamples, e.g., over a concentration range of more than 10⁵-fold,10⁶-fold, or 10⁷-fold.

The high sensitivity of the instruments, kits, and methods of theinvention allows the use of markers, e.g., biological markers, whichwere not previously useful because of a lack of sensitivity ofdetection. The high sensitivity of the instruments, kits, and methods ofthe invention also facilitate the establishment of new markers. Thereare numerous markers currently available which could be useful indetermining biological states, but are not currently of practical usebecause of current limitations in measuring their lower concentrationranges. In some cases, abnormally high levels of the marker aredetectable by current methods, but normal ranges are unknown. In somecases, abnormally high levels of the marker are detectable by currentmethods, but normal ranges have not been established. In some cases,upper normal ranges of the marker are detectable, but not lower normalranges, or levels below normal. In some cases, e.g., markers of canceror infection, any level of the marker can indicate the presence of abiological state, and enhancing sensitivity of detection is an advantagefor early diagnosis. In some cases, the rate of change, or lack ofchange, in the concentration of a marker over multiple time pointsprovides the most useful information, but present methods of analysis donot permit time point sampling in the early stages of a condition whenit is typically most treatable. In some cases, the marker can bedetected at clinically useful levels only through the use of cumbersomemethods that are not practical or useful in a clinical setting, such asmethods that require complex sample treatment and time-consuminganalysis. In addition, there are potential markers of biological stateswith sufficiently low concentration that their presence remainsextremely difficult or impossible to detect by current methods.

The analytical methods and compositions of the present invention providelevels of sensitivity, precision, and robustness that allow thedetection of markers for biological states at concentrations at whichthe markers have been previously undetectable, thus allowing the“repurposing” of such markers from confirmatory markers, or markersuseful only in limited research settings, to diagnostic, prognostic,treatment-directing, or other types of markers useful in clinicalsettings and/or in large scale clinical settings, including clinicaltrials. Such methods allow the determination of normal and abnormalranges for such markers.

The markers thus repurposed can be used for, e.g., detection of normalstate (normal ranges), detection of responder/non-responder (e.g., to atreatment, such as administration of a drug); detection of early diseaseor pathological occurrence (e.g., early detection of cancer, earlydetection of cardiac ischemia); disease staging (e.g., cancer); diseasemonitoring (e.g., diabetes monitoring, monitoring for cancer recurrenceafter treatment); study of disease mechanism; and study of treatmenttoxicity, such as toxicity of drug treatments.

The invention thus provides methods and compositions for the sensitivedetection of markers, and further methods of establishing values fornormal and abnormal levels of markers. In further embodiments, theinvention provides methods of diagnosis, prognosis, and/or treatmentselection based on values established for the markers. The inventionalso provides compositions for use in such methods, e.g., detectionreagents for the ultrasensitive detection of markers.

II. MOLECULES FOR SENSITIVE DETECTION BY THE METHODS AND COMPOSITIONS OFTHE INVENTION

The instruments, kits and methods of the invention can be used for thesensitive detection and determination of concentration of a number ofdifferent types of single molecules. In particular, the instruments,kits, and methods are useful in the sensitive detection anddetermination of concentration of markers of biological states.“Detection of a single molecule,” as that term is used herein, refers toboth direct and indirect detection. For example, a single molecule maybe labeled with a fluorescent label, and the molecule-label complexdetected in the instruments described herein. Alternatively, a singlemolecule may be labeled with a fluorescent label, then the fluorescentlabel is detached from the single molecule, and the label detected inthe instruments described herein. The term detection of a singlemolecule encompasses both forms of detection.

A. General

Examples of molecules which can be detected using the analyzer andrelated methods of the present invention include: biopolymers such asproteins, nucleic acids, carbohydrates, and small molecules, bothorganic and inorganic. In particular, the instruments, kits, and methodsdescribed herein are useful in the detection of single molecules ofproteins and small molecules in biological samples, and thedetermination of concentration of such molecules in the sample.

The terms “protein,” “polypeptide,” “peptide,” and “oligopeptide,” areused interchangeably herein and include any composition that includestwo or more amino acids joined together by a peptide bond. It may beappreciated that polypeptides can contain amino acids other than the 20amino acids commonly referred to as the 20 naturally occurring aminoacids. Also, polypeptides can include one or more amino acids, includingthe terminal amino acids, which are modified by any means known in theart (whether naturally or non-naturally). Examples of polypeptidemodifications include e.g., by glycosylation, orother-post-translational modification. Modifications which may bepresent in polypeptides of the present invention, include, but are notlimited to, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a polynucleotide or polynucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

The molecules detected by the present instruments, kits, and methods maybe free or may be part of a complex, e.g., an antibody-antigen complex,or more generally a protein-protein complex, e.g., complexes of troponinor prostate specific antigen (PSA). One of skill in the art willappreciate that when referring to proteins, the present invention candetect fragments, polypeptides, mutants, variants or complexes thereof.

B. Markers of Biological States

In some embodiments, the invention provides compositions and methods forthe sensitive detection of biological markers, and for the use of suchmarkers in diagnosis, prognosis, and/or determination of methods oftreatment.

Markers of the present invention may be, for example, any compositionand/or molecule or a complex of compositions and/or molecules that isassociated with a biological state of an organism (e.g., a conditionsuch as a disease or a non-disease state). A marker can be, for example,a small molecule, a polypeptide, a nucleic acid, such as DNA and RNA, alipid, such as a phospholipid or a micelle, a cellular component such asa mitochondrion or chloroplast, etc. Markers contemplated by the presentinvention can be previously known or unknown. For example, in someembodiments, the methods herein may identify novel polypeptides that canbe used as markers for a biological state of interest or condition ofinterest, while in other embodiments, known polypeptides are identifiedas markers for a biological state of interest or condition. Using thesystems of the invention it is possible that one can observe thosemarkers, e.g., polypeptides with high potential use in determining thebiological state of an organism, but that are only present at lowconcentrations, such as those “leaked” from diseased tissue. Other highpotentially useful markers or polypeptides may be those that are relatedto the disease, for instance, those that are generated in the tumor-hostenvironment. Any suitable marker that provides information regarding abiological state may be used in the methods and compositions of theinvention. A “marker,” as that term is used herein, includes anymolecule that may be detected in a sample from an organism and whosedetection or quantitation provides information about the biologicalstate of the organism.

Biological states include but are not limited to phenotypic states;conditions affecting an organism; states of development; age; health;pathology; disease detection, process, or staging; infection; toxicity;or response to chemical, environmental, or drug factors (such as drugresponse phenotyping, drug toxicity phenotyping, or drug effectivenessphenotyping).

The term “organism” as used herein refers to any living being comprisedof a least one cell. An organism can be as simple as a one cell organismor as complex as a mammal. An organism of the present invention ispreferably a mammal. Such mammal can be, for example, a human or ananimal such as a primate (e.g., a monkey, chimpanzee, etc.), adomesticated animal (e.g., a dog, cat, horse, etc.), farm animal (e.g.,goat, sheep, pig, cattle, etc.), or laboratory animal (e.g., mouse, rat,etc.). Preferably, an organism is a human.

In some embodiments, the methods and compositions of the invention aredirected to classes of markers, e.g., cytokines, growth factors,oncology markers, markers of inflammation, endocrine markers, autoimmunemarkers, thyroid markers, cardiovascular markers, markers of diabetes,markers of infectious disease, neurological markers, respiratorymarkers, gastrointestinal markers, musculoskeletal markers,dermatological disorders, and metabolic markers.

Table 1 provides examples of these classes of markers that have beenmeasured by the methods and compositions of the invention, and providesexemplary concentrations of the markers detected by the methods andcompositions of the invention and number of particles that are countedby the single particle analyzer system of the invention for theparticular marker.

TABLE 1 CLASSES OF MARKERS AND EXEMPLARY MARKERS IN THE CLASSES MolarConc. Molecules Cytokines IL-12 p70 2.02 × 10⁻¹⁴ 6.09 × 10⁺⁵ IL-10 5.36× 10⁻¹⁴ 1.61 × 10⁺⁶ IL-1 alpha 5.56 × 10⁻¹⁴ 1.67 × 10⁺⁶ IL-3 5.85 ×10⁻¹⁴ 1.76 × 10⁺⁶ IL-12 p40 6.07 × 10⁻¹⁴ 1.83 × 10⁺⁶ IL-1ra 6.12 × 10⁻¹⁴1.84 × 10⁺⁶ IL-12 8.08 × 10⁻¹⁴ 2.44 × 10⁺⁶ IL-6 9.53 × 10⁻¹⁴ 2.87 × 10⁺⁶IL-4 1.15 × 10⁻¹³ 3.47 × 10⁺⁶ IL-18 1.80 × 10⁻¹³ 5.43 × 10⁺⁶ IP-10 1.88× 10⁻¹³ 1.13 × 10⁺⁷ IL-5 1.99 × 10⁻¹³ 5.98 × 10⁺⁶ Eotaxin 2.06 × 10⁻¹³1.24 × 10⁺⁷ IL-16 3.77 × 10⁻¹³ 1.14 × 10⁺⁷ MIG 3.83 × 10⁻¹³ 1.15 × 10⁺⁷IL-8 4.56 × 10⁻¹³ 1.37 × 10⁺⁷ IL-17 5.18 × 10⁻¹³ 1.56 × 10⁺⁷ IL-7 5.97 ×10⁻¹³ 1.80 × 10⁺⁷ IL-15 6.13 × 10⁻¹³ 1.84 × 10⁺⁷ IL-13 8.46 × 10⁻¹³ 2.55× 10⁺⁷ IL-2R (soluble) 8.89 × 10⁻¹³ 2.68 × 10⁺⁷ IL-2 8.94 × 10⁻¹³ 2.69 ×10⁺⁷ LIF/HILDA 9.09 × 10⁻¹³ 5.47 × 10⁺⁷ IL-1 beta 1.17 × 10⁻¹² 3.51 ×10⁺⁷ Fas/CD95/Apo-1 1.53 × 10⁻¹² 9.24 × 10⁺⁷ MCP-1 2.30 × 10⁻¹² 6.92 ×10⁺⁷ Oncology EGF 4.75 × 10⁻¹⁴ 2.86 × 10⁺⁶ TNF-alpha 6.64 × 10⁻¹⁴ 8.00 ×10⁺⁶ PSA (3rd generation) 1.15 × 10⁻¹³ 6.92 × 10⁺⁶ VEGF 2.31 × 10⁻¹³6.97 × 10⁺⁶ TGF-beta1 2.42 × 10⁻¹³ 3.65 × 10⁺⁷ FGFb 2.81 × 10⁻¹³ 1.69 ×10⁺⁷ TRAIL 5.93 × 10⁻¹³ 3.57 × 10⁺⁷ TNF-RI (p55) 2.17 × 10⁻¹² 2.62 ×10⁺⁸ Inflammation ICAM-1 (soluble) 8.67 × 10⁻¹⁵ 5.22 × 10⁺⁴ RANTES 6.16× 10⁻¹⁴ 3.71 × 10⁺⁶ MIP-2 9.92 × 10⁻¹⁴ 2.99 × 10⁺⁶ MIP-1 beta 1.98 ×10⁻¹³ 5.97 × 10⁺⁶ MIP-1 alpha 2.01 × 10⁻¹³ 6.05 × 10⁺⁶ MMP-3 1.75 ×10⁻¹² 5.28 × 10⁺⁷ Endocrinology 17 beta-Estradiol (E2) 4.69 × 10⁻¹⁴ 2.82× 10⁺⁶ DHEA 4.44 × 10⁻¹³ 2.67 × 10⁺⁷ ACTH 1.32 × 10⁻¹² 7.96 × 10⁺⁷Gastrin 2.19 × 10⁻¹² 1.32 × 10⁺⁸ Growth Hormone (hGH) 2.74 × 10⁻¹² 1.65× 10⁺⁸ Autoimmune GM-CSF 1.35 × 10⁻¹³ 8.15 × 10⁺⁶ C-Reactive Protein(CRP) 3.98 × 10⁻¹³ 2.40 × 10⁺⁷ G-CSF 1.76 × 10⁻¹² 1.06 × 10⁺⁸ ThyroidCyclic AMP 9.02 × 10⁻¹⁵ 5.43 × 10⁺⁵ Calcitonin 3.25 × 10⁻¹⁴ 1.95 × 10⁺⁶Parathyroid Hormone (PTH) 1.56 × 10⁻¹³ 9.37 × 10⁺⁶ CardiovascularB-Natriuretic Peptide 2.86 × 10⁻¹³ 1.72 × 10⁺⁷ NT-proBNP 2.86 × 10⁻¹²8.60 × 10⁺⁷ C-Reactive Protein, HS 3.98 × 10⁻¹³ 2.40 × 10⁺⁷Beta-Thromboglobulin (BTG) 5.59 × 10⁻¹³ 3.36 × 10⁺⁷ Diabetes C-Peptide2.41 × 10⁻¹⁵ 1.45 × 10⁺⁵ Leptin 1.89 × 10⁻¹³ 1.14 × 10⁺⁷ Infectious Dis.IFN-gamma 2.08 × 10⁻¹³ 1.25 × 10⁺⁷ IFN-alpha 4.55 × 10⁻¹³ 2.74 × 10⁺⁷Metabolism Bio-Intact PTH (1-84) 1.59 × 10⁻¹² 1.44 × 10⁺⁸ PTH 1.05 ×10⁻¹³ 9.51 × 10⁺⁶

1. Cytokines

For both research and diagnostics, cytokines are useful as markers of anumber of conditions, diseases, pathologies, and the like, and thecompositions and methods of the invention include labels for detectionand quantitation of cytokines and methods using such labels to determinenormal and abnormal levels of cytokines, as well as methods ofdiagnosis, prognosis, and/or determination of treatment based on suchlevels.

There are currently over 100 cytokines/chemokines whose coordinate ordiscordant regulation is of clinical interest. In order to correlate aspecific disease process with changes in cytokine levels, the idealapproach requires analyzing a sample for a given cytokine, or multiplecytokines, with high sensitivity. Exemplary cytokines that are presentlyused in marker panels and that may be used in methods and compositionsof the invention include, but are not limited to, BDNF, CREB pS133, CREBTotal, DR-5, EGF, ENA-78, Eotaxin, Fatty Acid Binding Protein,FGF-basic, granulocyte colony-stimulating factor (G-CSF), GCP-2,Granulocyte-macrophage Colony-stimulating Factor GM-CSF (GM-CSF),growth-related oncogene-keratinocytes (GRO-KC), HGF, ICAM-1, IFN-alpha,IFN-gamma, the interleukins IL-10, IL-11, IL-12, IL-12 p40, IL-12p40/p70, IL-12 p70, IL-13, IL-15, IL-16, IL-17, IL-18, IL-1alpha,IL-1beta, IL-1ra, IL-1ra/IL-1F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, interferon-inducible protein (10 IP-10), JE/MCP-1,keratinocytes (KC), KC/GROa, LIF, Lymphotacin, M-CSF, monocytechemoattractant protein-1 (MCP-1), MCP-1(MCAF), MCP-3, MCP-5, MDC, MIG,macrophage inflammatory (MIP-1 alpha), MIP-1 beta, MIP-1 gamma, MIP-2,MIP-3 beta, OSM, PDGF-BB, regulated upon activation, normal T cellexpressed and secreted (RANTES), Rb (pT821), Rb (total), Rb pSpT249/252,Tau (pS214), Tau (pS396), Tau (total), Tissue Factor, tumor necrosisfactor-alpha (TNF-alpha), TNF-beta, TNF-RI, TNF-RII, VCAM-1, and VEGF.In some embodiments, the cytokine is IL-12p70, IL-10, IL-1 alpha, IL-3,IL-12 p40, IL-1ra, IL-12, IL-6, IL-4, IL-18, IL-10, IL-5, eotaxin,IL-16, MIG, IL-8, IL-17, IL-7, IL-15, IL-13, IL-2R (soluble), IL-2,LIF/HILDA, IL-1 beta, Fas/CD95/Apo-1, or MCP-1.

2. Growth factors

Growth factors include EGF Ligands such as Amphiregulin, LRIG3,Betacellulin, Neuregulin-1/NRG1, EGF, Neuregulin-3/NRG3, Epigen,TGF-alpha, Epiregulin, TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, LRIG1; EGFR/ErbB Receptor Family such as EGF R, ErbB3, ErbB2, ErbB4; FGF Familysuch as FGF LigandsFGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16,FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9,FGF-22, FGF-10, FGF-23, FGF-11, KGF/FGF-7, FGF Receptors FGF R1-4, FGFR3, FGF R1, FGF R4, FGF R2, FGF R5, FGF Regulators FGF-BP; the HedgehogFamily Desert Hedgehog, Sonic Hedgehog, Indian Hedgehog; HedgehogRelated Molecules & Regulators BOC, GLI-3, CDO, GSK-3 alpha/beta, DISP1,GSK-3 alpha, Gas1, GSK-3 beta, GLI-1, Hip, GLI-2; the IGF Family IGFLigands IGF-I, IGF-II, IGF-I Receptor (CD221)IGF-I R, and IGF BindingProtein (IGFBP) Family ALS, IGFBP-5, CTGF/CCN2, IGFBP-6, Cyr61/CCN1,IGFBP-L1, Endocan, IGFBP-rp1/IGFBP-7, IGFBP-1, IGFBP-rP10, IGFBP-2,NOV/CCN3, IGFBP-3, WISP-1/CCN4, IGFBP-4; Receptor Tyrosine Kinases Ax1,FGF R4, C1q R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF-I R,EGF, R IGF-II R, Eph, INSRR, EphA1, Insulin R/CD220, EphA2, M-CSF R,EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R alpha, EphA7,PDGF R beta, EphA8, Ret, EphB1, RTK-like Orphan Receptor 1/ROR1, EphB2,RTK-like Orphan Receptor 2/ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1,EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF, R1-4 VEGF R,FGF R1, VEGF R1/Flt-1, FGF R2, VEGF R2/KDR/Flk-1, FGF R3, VEGF R3/Flt-4;Proteoglycans & Regulators Proteoglycans Aggrecan, Mimecan, Agrin,NG2/MCSP, Biglycan, Osteoadherin, Decorin, Podocan, DSPG3,delta-Sarcoglycan, Endocan, Syndecan-1/CD138, Endoglycan, Syndecan-2,Endorepellin/Perlecan, Syndecan-3, Glypican 2, Syndecan-4, Glypican 3,Testican 1/SPOCK1, Glypican 5, Testican 2/SPOCK2, Glypican 6, Testican3/SPOCK3, Lumican, Versican, Proteoglycan Regulators, ArylsulfataseA/ARSA, Glucosamine (N-acetyl)-6-Sulfatase/GNS, Exostosin-like 2/EXTL2,HS6ST2, Exostosin-like 3/EXTL3, Iduronate 2-Sulfatase/IDS, Ga1NAc4S-6ST;SCF, Flt-3 Ligand & M-CSF Flt-3, M-CSF R, Flt-3 Ligand, SCF, M-CSF, SCFR/c-kit; TGF-beta Superfamily (same as listed for inflammatory markers);VEGF/PDGF Family Neuropilin-1, P1GF, Neuropilin-2, P1GF-2, PDGF, VEGF,PDGF R alpha, VEGF-B, PDGF R beta, VEGF-C, PDGF-A, VEGF-D, PDGF-AB, VEGFR, PDGF-B, VEGF R1/Flt-1, PDGF-C, VEGF R2/KDR/Flk-1, PDGF-D, VEGFR3/Flt-4; Wnt-related Molecules Dickkopf Proteins & Wnt InhibitorsDkk-1, Dkk-4, Dkk-2, Soggy-1, Dkk-3, WIF-1 Frizzled & Related ProteinsFrizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1,Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7,MFRP; Wnt Ligands Wnt-1, Wnt-8a, Wnt-2b, Wnt-8b, Wnt-3a, Wnt-9a, Wnt-4,Wnt-9b, Wnt-5a, Wnt-10a, Wnt-5b, Wnt-10b, Wnt-7a, Wnt-11, Wnt-7b; OtherWnt-related Molecules APC, Kremen-2, Axin-1, LRP-1, beta-Catenin, LRP-6,Dishevelled-1, Norrin, Dishevelled-3, PKC beta 1, Glypican 3, Pygopus-1,Glypican 5, Pygopus-2, GSK-3 alpha/beta, R-Spondin 1, GSK-3 alpha,R-Spondin 2, GSK-3 beta, R-Spondin 3, ICAT, RTK-like Orphan Receptor1/ROR1, Kremen-1, RTK-like Orphan Receptor 2/ROR, and Other GrowthFactors CTGF/CCN2, beta-NGF, Cyr61/CCN1, Norrin, DANCE, NOV/CCN3,EG-VEGF/PK1, Osteocrin, Hepassocin, PD-ECGF, HGF, Progranulin, LECT2,Thrombopoietin, LEDGF, WISP-1/CCN4.

3. Markers of Inflammation

Markers of inflammation include ICAM-1, RANTES, MIP-2, MIP-1-beta,MIP-1-alpha, and MMP-3. Further markers of inflammation include Adhesionmolecules such as the integrins α1β1, α2β1, α3β1, α4β1, α5β1, α6β1,α7β1, α8β1, α9β1, αVβ1, α4β7, α6β4, αDβ2, αLβ2, αMβ2, αVβ3, αVβ5, αVβ6,αVβ8, αXβ2, αIIbβ3, αIELbβ7, beta-2 integrin, beta-3 integrin, beta-2integrin, beta-4 integrin, beta-5 integrin, beta-6 integrin, beta-7integrin, beta-8 integrin, alpha-1 integrin, alpha-2 integrin, alpha-3integrin, alpha-4 integrin, alpha-5 integrin, alpha-6 integrin, alpha-7integrin, alpha-8 integrin, alpha-9 integrin, alpha-D integrin, alpha-Lintegrin, alpha-M integrin, alpha-V integrin, alpha-X integrin,alpha-IIb integrin, alphaIELb integrin; Integrin-associated Moleculessuch as Beta IG-H3, Melusin, CD47, MEPE, CD151, Osteopontin,IBSP/Sialoprotein II, RAGE, IGSF8; Selectins such as E-Selectin,P-Selectin, L-Selectin; Ligands such as CD34, GlyCAM-1, MadCAM-1,PSGL-1, vitronectic, vitronectin receptor, fibronectin, vitronectin,collagen, laminin, ICAM-1, ICAM-3, BL-CAM, LFA-2, VCAM-1, NCAM, PECAM.Further markers of inflammation include Cytokines such as IFN-α, IFN-β,IFN-ε, -κ, -τ, and -ζ, IFN-ω, IFN-γ, IL29, IL28A and IL28B, IL-1, IL-1αand β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21,IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,TCCR/WSX-1. Further markers of inflammation include cytokine receptorssuch as Common beta chain, IL-3 R alpha, IL-3 R beta, GM-CSF R, IL-5 Ralpha, Common gamma Chain/IL-2 R gamma, IL-2 R alpha, IL-9 R, IL-2 Rbeta, IL-4 R, IL-21 R, IL-15 R alpha, IL-7 R alpha/CD127, IL-1ra/IL-1F3,IL-1 R8, IL-1 RI, IL-1 R9, IL-1 R11, IL-18 R alpha/IL-1 R5, IL-1 R3/IL-1R AcP, IL-18 R beta/IL-1 R7, IL-1 R4/ST2 SIGIRR, IL-R6/IL-1 R rp2, IL-11R alpha, IL-31 RA, CNTF R alpha, Leptin R, G-CSF R, LIF R alpha, IL-6 R,OSM R beta, IFN-alpha/beta R1, IFN-alpha/beta R2, IFN-gamma R1,IFN-gamma R2, IL-10 R alpha, IL-10 R beta, IL-20 R alpha, IL-20 R beta,IL-22 R, IL-17 R, IL-17 RD, IL-17 RC, IL-17B R, IL-13 R alpha 2, IL-23R, IL-12 R beta 1, IL-12 R beta 2, TCCR/WSX-1, IL-13 R alpha 1. Furthermarkers of inflammation include Chemokines such as CCL-1, CCL-2, CCL-3,CCL-4, CCL-5, CCL-6, CCL-7, CCL-8, CCL-9, CCL-10, CCL-11, CCL-12,CCL-13, CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-20, CCL-21,CCL-22, CCL-23, CCL-24, CCL-25, CCL-26, CCL-27, CCL-28, MCK-2, MIP-2,CINC-1, CINC-2, KC, CINC-3, LIX, GRO, Thymus Chemokine-1, CXCL-1,CXCL-2, CXCL-3, CXCL-4, CXCL-5, CXCL-6, CXCL-7, CXCL-8, CXCL-9, CXCL-10,CXCL-11, CXCL-12, CXCL-13, CXCL-14, CXCL-15, CXCL-16, CXCL-17, XCL1,XCL2, Chemerin. Further markers of inflammation include chemokinereceptors such as CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7,CCR-8, CCR-9, CCR-10, CXCR3, CXCR6, CXCR4, CXCR1, CXCR5, CXCR2, ChemR23. Further markers of inflammation include Tumor necrosis factors(TNFs), such as TNF-alpha, 4-1BB Ligand/TNFSF9, LIGHT/TNFSF14,APRIL/TNFSF13, Lymphotoxin, BAFF/TNFSF13B, Lymphotoxin beta/TNFSF3, CD27Ligand/TNFSF7, OX40 Ligand/TNFSF4, CD30 Ligand/TNFSF8, TL1A/TNFSF15,CD40 Ligand/TNFSF5, TNF-alpha/TNFSF1A, EDA, TNF-beta/TNFSF1B, EDA-A2,TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18,TWEAK/TNFSF12. Further markers of inflammation include TNF SuperfamilyReceptors such as 4-1BB/TNFRSF9, NGF R/TNFRSF16, BAFF R/TNFRSF13C,Osteoprotegerin/TNFRSF11B, BCMA/TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7,RANK/TNFRSF11A, CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF5,TACI/TNFRSF13B, DcR3/TNFRSF6B, TNF R1/TNFRSF1A, DcTRAIL R1/TNFRSF23, TNFR11/TNFRSF1B, DcTRAIL R2/TNFRSF22, TRAIL R1/TNFRSF100A, DR3/TNFRSF25,TRAIL R2/TNFRSF10B, DR6/TNFRSF21, TRAIL R3/TNFRSF10C, EDAR, TRAILR4/TNFRSF10D, Fas/TNFRSF6, TROY/TNFRSF19, GITR/TNFRSF18, TWEAKR/TNFRSF12, HVEM/TNFRSF14, XEDAR. Further markers of inflammationinclude TNF Superfamily Regulators such as FADD, TRAF-2, RIP1, TRAF-3,TRADD, TRAF-4, TRAF-1, TRAF-6. Further markers of inflammation includeAcute-phase reactants and acute phase proteins. Further markers ofinflammation include TGF-beta superfamily ligands such as Activins,Activin A, Activin B, Activin AB, Activin C, BMPs (Bone MorphogeneticProteins), BMP-2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4,BMP-10, BMP-5, BMP-15/GDF-9B, BMP-6, Decapentaplegic,Growth/Differentiation Factors (GDFs), GDF-1, GDF-8, GDF-3, GDF-9 GDF-5,GDF-11, GDF-6, GDF-15, GDF-7, GDNF Family Ligands, Artemin, Neurturin,GDNF, Persephin, TGF-beta, TGF-beta, TGF-beta 3, TGF-beta 1, TGF-beta 5,LAP (TGF-beta 1), Latent TGF-beta bp1, Latent TGF-beta 1, LatentTGF-beta bp2, TGF-beta 1.2, Latent TGF-beta bp4, TGF-beta 2, Lefty,MIS/AMH, Lefty-1, Nodal, Lefty-A, Activin RIA/ALK-2, GFR alpha-1/GDNF Ralpha-1, Activin RIB/ALK-4, GFR alpha-2/GDNF R alpha-2, Activin RIIA,GFR alpha-3/GDNF R alpha-3, Activin RIIB, GFR alpha-4/GDNF R alpha-4,ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TGF-beta R1/ALK-5,BMPR-IB/ALK-6, TGF-beta RII, BMPR-II, TGF-beta RIIb, Endoglin/CD105,TGF-beta RIII. Further markers of inflammation include TGF-betasuperfamily Modulators such as Amnionless, NCAM-1/CD56, BAMBI/NMA,Noggin, BMP-1/PCP, NOMO, Caronte, PRDC, Cerberus 1, SKI, Chordin, Smad1,Chordin-Like 1, Smad2, Chordin-Like 2, Smad3, COCO, Smad4, CRIM1, Smad5,Cripto, Smad7, Crossveinless-2, Smad8, Cryptic, SOST, DAN, LatentTGF-beta bp1, Decorin, Latent TGF-beta bp2, FLRG, Latent TGF-beta bp4,Follistatin, TMEFF1/Tomoregulin-1, Follistatin-like 1, TMEFF2,GASP-1/WFIKKNRP, TSG, GASP-2/WFIKKN, TSK, Gremlin, Vasorin. Furthermarkers of inflammation include EGF Ligands such as Amphiregulin, LRIG3,Betacellulin, Neuregulin-1/NRG1, EGF, Neuregulin-3/NRG3, Epigen,TGF-alpha, Epiregulin, TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, LRIG1.Further markers of inflammation include EGF R/ErbB Receptor Family, suchas EGF R, ErbB3, ErbB2, ErbB4. Further markers of inflammation includeFibrinogen. Further markers of inflammation include SAA. Further markersof inflammation include glial markers, such as alpha.1-antitrypsin,C-reactive protein (CRP), alpha.2-macroglobulin, glial fibrillary acidicprotein (GFAP), Mac-1, F4/80. Further markers of inflammation includemyeloperoxidase. Further markers of inflammation include Complementmarkers such as C3d, C1q, C5, C4d, C4 bp, and C5a-C9. Further markers ofinflammation include Major histocompatibility complex (MHC)glycoproteins, such as HLA-DR and HLA-A,D,C. Further markers ofinflammation include Microglial markers, such as CR3 receptor, MHC I,MHC II, CD 31, CD11a, CD11b, CD11c, CD68, CD45RO, CD45RD, CD18, CD59,CR4, CD45, CD64, and CD44. Further markers of inflammation includealpha.2 macroglobulin receptor, Fibroblast growth factor, Fc gamma RI,Fc gamma RII, CD8, LCA (CD45), CD18, CD59, Apo J, clusterin, type 2plasminogen activator inhibitor, CD44, Macrophage colony stimulatingfactor receptor, MRP14, 27E10, 4-hydroxynonenal-protein conjugates,I.kappa.B, NF.kappa.B, cPLA.sub.2, COX-2, Matrix metalloproteinases,Membrane lipid peroxidation, and ATPase activity. HSPC228, EMP1, CDC42,TLE3, SPRY2, p40BBP, HSPC060 or NAB2, or a down-regulation of HSPA1A,HSPA1B, MAPRE2 and OAS1 expression, TACE/ADAM17, alpha-1-AcidGlycoprotein, Angiopoietin-1, MIF, Angiopoietin-2, CD14, beta-Defensin2, MMP-2, ECF-L/CHI3L3, MMP-7, EGF, MMP-9, EMAP-II, MSP, EN-RAGE, NitricOxide, Endothelin-1, Osteoactivin/GPNMB, FPR1, PDGF, FPRL1, Pentraxin3/TSG-14, FPRL2, Gas6, PLUNC, GM-CSF, RAGE, S100A10, S100A8, S100A9,HIF-1 alpha, Substance P, TFPI, TGF-beta 1, TIMP-1, TIMP-2, TIMP-3,TIMP-4, TLR4, LBP, TREM-1, Leukotriene A4, Hydrolase TSG-6, Lipocalin-1,uPA, M-CSF, and VEGF.

4. Miscellaneous Markers

Oncology markers include EGF, TNF-alpha, PSA, VEGF, TGF-beta1, FGFb,TRAIL, and TNF-R1 (p55).

Markers of endocrine function include 17 beta-estradiol (E2), DHEA,ACTH, gastrin, and growth hormone (hGH).

Markers of autoimmunity include GM-CSF, C-Reactive Protein, and G-CSF.

Markers of thyroid function include cyclicAMP, calcitonin, andparathyroid hormone.

Cardiovascular markers include cardiac troponin I, cardiac troponin T,B-natriuretic peptide, NT-proBNP, C-Reactive Protein HS, andbeta-thromboglobulin.

Markers of diabetes include C-peptide and leptin.

Markers of infectious disease include IFN-gamma and IFN-alpha.

Markers of metabolism include Bio-intact PTH (1-84) and PTH.

5. Markers of Biological States

Markers may indicate the presence of a particular phenotypic state ofinterest. Examples of phenotypic states include, phenotypes resultingfrom an altered environment, drug treatment, genetic manipulations ormutations, injury, change in diet, aging, or any other characteristic(s)of a single organism or a class or subclass of organisms.

In some embodiments, a phenotypic state of interest is a clinicallydiagnosed disease state. Such disease states include, for example,cancer, cardiovascular disease, inflammatory disease, autoimmunedisease, neurological disease, infectious disease and pregnancy relateddisorders. Alternatively, states of health can be detected usingmarkers.

Cancer phenotypes are included in some aspects of the invention.Examples of cancer include, but are not limited to: breast cancer, skincancer, bone cancer, prostate cancer, liver cancer, lung cancer, braincancer, cancer of the larynx, gallbladder, pancreas, rectum,parathyroid, thyroid, adrenal, neural tissue, head and neck, colon,stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinomaof both ulcerating and papillary type, metastatic skin carcinoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant celltumor, small-cell lung tumor, non-small cell lung carcinoma gallstones,islet cell tumor, primary brain tumor, acute and chronic lymphocytic andgranulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullarycarcinoma, pheochromocytoma, mucosal neuromas, intestinalganglloneuromas, hyperplastic corneal nerve tumor, marfanoid habitustumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

The present invention provides methods to detect cancers. In someembodiments, the cancer comprises Acute Lymphoblastic Leukemia. In otherembodiments, the cancer comprises Acute Myeloid Leukemia. In otherembodiments, the cancer comprises Adrenocortical Carcinoma. In otherembodiments, the cancer comprises an AIDS-Related Cancer. In otherembodiments, the cancer comprises AIDS-Related Lymphoma. In otherembodiments, the cancer comprises Anal Cancer. In other embodiments, thecancer comprises Appendix Cancer. In other embodiments, the cancercomprises Childhood Cerebellar Astrocytoma. In other embodiments, thecancer comprises Childhood Cerebral Astrocytoma. In other embodiments,the cancer comprises a Central Nervous System Atypical Teratoid/RhabdoidTumor. In other embodiments, the cancer comprises Basal Cell Carcinoma,or other Skin Cancer (Nonmelanoma). In other embodiments, the cancercomprises Extrahepatic Bile Duct Cancer. In other embodiments, thecancer comprises Bladder Cancer. In other embodiments, the cancercomprises Bone Cancer, such as Osteosarcoma or Malignant FibrousHistiocytoma. In other embodiments, the cancer comprises Brain StemGlioma. In other embodiments, the cancer comprises an Adult Brain Tumor.In other embodiments, the cancer comprises Brain Tumor comprisingCentral Nervous System Atypical Teratoid/Rhabdoid Tumor. In otherembodiments, the cancer comprises a Brain Tumor comprising CerebralAstrocytoma/Malignant Glioma. In other embodiments, the cancer comprisesa Craniopharyngioma Brain Tumor. In other embodiments, the cancercomprises a Ependymoblastoma Brain Tumor. In other embodiments, thecancer comprises a Ependymoma Brain Tumor. In other embodiments, thecancer comprises a Medulloblastoma Brain Tumor. In other embodiments,the cancer comprises a Medulloepithelioma Brain Tumor. In otherembodiments, the cancer comprises Brain Tumors including PinealParenchymal Tumors of Intermediate Differentiation. In otherembodiments, the cancer comprises Brain Tumors including SupratentorialPrimitive Neuroectodermal Tumors and Pineoblastoma. In otherembodiments, the cancer comprises a Brain Tumor including Visual Pathwayand Hypothalamic Glioma. In other embodiments, the cancer comprisesBrain and Spinal Cord Tumors. In other embodiments, the cancer comprisesBreast Cancer. In other embodiments, the cancer comprises BronchialTumors. In other embodiments, the cancer comprises Burkitt Lymphoma. Inother embodiments, the cancer comprises Carcinoid Tumor. In otherembodiments, the cancer comprises Gastrointestinal Carcinoid Tumor. Inother embodiments, the cancer comprises Carcinoma of Unknown PrimaryOrigin. In other embodiments, the cancer comprises Central NervousSystem Atypical Teratoid/Rhabdoid Tumor. In other embodiments, thecancer comprises Central Nervous System Embryonal Tumors. In otherembodiments, the cancer comprises Primary Central Nervous SystemLymphoma. In other embodiments, the cancer comprises CerebellarAstrocytoma. In other embodiments, the cancer comprises CerebralAstrocytoma/Malignant Glioma. In other embodiments, the cancer comprisesCervical Cancer. In other embodiments, the cancer comprises ChildhoodCancers. In other embodiments, the cancer comprises Chordoma. In otherembodiments, the cancer comprises Chronic Lymphocytic Leukemia. In otherembodiments, the cancer comprises Chronic Myelogenous Leukemia. In otherembodiments, the cancer comprises Chronic Myeloproliferative Disorders.In other embodiments, the cancer comprises Colon Cancer. In otherembodiments, the cancer comprises Colorectal Cancer. In otherembodiments, the cancer comprises Craniopharyngioma. In otherembodiments, the cancer comprises Cutaneous T-Cell Lymphoma, includingMycosis Fungoides and Sézary Syndrome. In other embodiments, the cancercomprises Central Nervous System Embryonal Tumors. In other embodiments,the cancer comprises Endometrial Cancer. In other embodiments, thecancer comprises Ependymoblastoma. In other embodiments, the cancercomprises Ependymoma. In other embodiments, the cancer comprisesEsophageal Cancer. In other embodiments, the cancer comprises the EwingFamily of Tumors. In other embodiments, the cancer comprisesExtracranial Germ Cell Tumor. In other embodiments, the cancer comprisesExtragonadal Germ Cell Tumor. In other embodiments, the cancer comprisesExtrahepatic Bile Duct Cancer. In other embodiments, the cancercomprises Intraocular Melanoma Eye Cancer. In other embodiments, thecancer comprises Retinoblastoma Eye Cancer. In other embodiments, thecancer comprises Gallbladder Cancer. In other embodiments, the cancercomprises Gastric (Stomach) Cancer. In other embodiments, the cancercomprises Gastrointestinal Carcinoid Tumor. In other embodiments, thecancer comprises Gastrointestinal Stromal Tumor (GIST). In otherembodiments, the cancer comprises Gastrointestinal Stromal Cell Tumor.In other embodiments, the cancer comprises Extracranial Germ Cell Tumor.In other embodiments, the cancer comprises Extragonadal Germ Cell Tumor.In other embodiments, the cancer comprises Ovarian Germ Cell Tumor. Inother embodiments, the cancer comprises Gestational Trophoblastic Tumor.In other embodiments, the cancer comprises Glioma. In other embodiments,the cancer comprises Brain Stem Glioma. In other embodiments, the cancercomprises Cerebral Astrocytoma Glioma. In other embodiments, the cancercomprises Visual Pathway or Hypothalamic Glioma. In other embodiments,the cancer comprises Hairy Cell Leukemia. In other embodiments, thecancer comprises Head and Neck Cancer. In other embodiments, the cancercomprises Hepatocellular (Liver) Cancer. In other embodiments, thecancer comprises Hodgkin Lymphoma. In other embodiments, the cancercomprises Hypopharyngeal Cancer. In other embodiments, the cancercomprises Intraocular Melanoma. In other embodiments, the cancercomprises Islet Cell Tumors (Endocrine Pancreas). In other embodiments,the cancer comprises Kaposi Sarcoma. In other embodiments, the cancercomprises Kidney (Renal Cell) Cancer. In other embodiments, the cancercomprises Laryngeal Cancer. In other embodiments, the cancer comprisesAcute Lymphoblastic Leukemia. In other embodiments, the cancer comprisesAcute Myeloid Leukemia. In other embodiments, the cancer comprisesChronic Lymphocytic Leukemia. In other embodiments, the cancer comprisesChronic Myelogenous Leukemia. In other embodiments, the cancer comprisesHairy Cell Leukemia. In other embodiments, the cancer comprises LipCancer. In other embodiments, the cancer comprises Oral Cavity Cancer.In other embodiments, the cancer comprises Primary Liver Cancer. Inother embodiments, the cancer comprises Non-Small Cell Lung Cancer. Inother embodiments, the cancer comprises Small Cell Lung Cancer. In otherembodiments, the cancer comprises AIDS-Related Lymphoma. In otherembodiments, the cancer comprises Burkitt Lymphoma. In otherembodiments, the cancer comprises Cutaneous T-Cell Lymphoma. In otherembodiments, the cancer comprises Mycosis Fungoides and Sezary Syndrome.In other embodiments, the cancer comprises Hodgkin Lymphoma. In otherembodiments, the cancer comprises Non-Hodgkin Lymphoma. In otherembodiments, the cancer comprises Primary Central Nervous SystemLymphoma. In other embodiments, the cancer comprises WaldenstrimMacroglobulinemia. In other embodiments, the cancer comprises MalignantFibrous Histiocytoma of Bone or Osteosarcoma. In other embodiments, thecancer comprises Medulloepithelioma. In other embodiments, the cancercomprises Melanoma. In other embodiments, the cancer comprisesIntraocular (Eye) Melanoma. In other embodiments, the cancer comprisesMerkel Cell Carcinoma. In other embodiments, the cancer comprisesMesothelioma. In other embodiments, the cancer comprises MetastaticSquamous Neck Cancer with Occult Primary. In other embodiments, thecancer comprises Mouth Cancer. In other embodiments, the cancercomprises Multiple Endocrine Neoplasia Syndrome. In other embodiments,the cancer comprises Multiple Myeloma/Plasma Cell Neoplasm. In otherembodiments, the cancer comprises Mycosis Fungoides. In otherembodiments, the cancer comprises Myelodysplastic Syndromes. In otherembodiments, the cancer comprises Myelodysplastic or MyeloproliferativeDiseases. In other embodiments, the cancer comprises Chronic MyelogenousLeukemia. In other embodiments, the cancer comprises Acute MyeloidLeukemia. In other embodiments, the cancer comprises Multiple Myeloma.In other embodiments, the cancer comprises Chronic MyeloproliferativeDisorders. In other embodiments, the cancer comprises Nasal Cavity orParanasal Sinus Cancer. In other embodiments, the cancer comprisesNasopharyngeal Cancer. In other embodiments, the cancer comprisesNasopharyngeal Cancer. In other embodiments, the cancer comprisesNeuroblastoma. In other embodiments, the cancer comprises Non-HodgkinLymphoma. In other embodiments, the cancer comprises Non-Small Cell LungCancer. In other embodiments, the cancer comprises Oral Cancer. In otherembodiments, the cancer comprises Oral Cavity Cancer. In otherembodiments, the cancer comprises Oropharyngeal Cancer. In otherembodiments, the cancer comprises Osteosarcoma. In other embodiments,the cancer comprises Malignant Fibrous Histiocytoma of Bone. In otherembodiments, the cancer comprises Ovarian Cancer. In other embodiments,the cancer comprises Ovarian Epithelial Cancer. In other embodiments,the cancer comprises Ovarian Germ Cell Tumor. In other embodiments, thecancer comprises Ovarian Low Malignant Potential Tumor. In otherembodiments, the cancer comprises Pancreatic Cancer. In otherembodiments, the cancer comprises Islet Cell Tumor Pancreatic Cancer. Inother embodiments, the cancer comprises Papillomatosis. In otherembodiments, the cancer comprises Paranasal Sinus Cancer. In otherembodiments, the cancer comprises Nasal Cavity Cancer. In otherembodiments, the cancer comprises Parathyroid Cancer. In otherembodiments, the cancer comprises Penile Cancer. In other embodiments,the cancer comprises Pharyngeal Cancer. In other embodiments, the cancercomprises Pheochromocytoma. In other embodiments, the cancer comprisesPineal Parenchymal Tumors of Intermediate Differentiation. In otherembodiments, the cancer comprises Pineoblastoma or SupratentorialPrimitive Neuroectodermal Tumors. In other embodiments, the cancercomprises Pituitary Tumor. In other embodiments, the cancer comprisesPlasma Cell Neoplasm/Multiple Myeloma. In other embodiments, the cancercomprises Pleuropulmonary Blastoma. In other embodiments, the cancercomprises Primary Central Nervous System Lymphoma. In other embodiments,the cancer comprises Prostate Cancer. In other embodiments, the cancercomprises Rectal Cancer. In other embodiments, the cancer comprisesRenal Cell (Kidney) Cancer. In other embodiments, the cancer comprisesRenal Pelvis and Ureter, Transitional Cell Cancer. In other embodiments,the cancer comprises Respiratory Tract Carcinoma Involving the NUT Geneon Chromosome 15. In other embodiments, the cancer comprisesRetinoblastoma. In other embodiments, the cancer comprisesRhabdomyosarcoma. In other embodiments, the cancer comprises SalivaryGland Cancer. In other embodiments, the cancer comprises Sarcoma of theEwing Family of Tumors. In other embodiments, the cancer comprisesKaposi Sarcoma. In other embodiments, the cancer comprises Soft TissueSarcoma. In other embodiments, the cancer comprises Uterine Sarcoma. Inother embodiments, the cancer comprises Sezary Syndrome. In otherembodiments, the cancer comprises Nonmelanoma Skin Cancer. In otherembodiments, the cancer comprises Melanoma Skin Cancer. In otherembodiments, the cancer comprises Merkel Cell Skin Carcinoma. In otherembodiments, the cancer comprises Small Cell Lung Cancer. In otherembodiments, the cancer comprises Small Intestine Cancer. In otherembodiments, the cancer comprises Squamous Cell Carcinoma, e.g.,Nonmelanoma Skin Cancer. In other embodiments, the cancer comprisesMetastatic Squamous Neck Cancer with Occult Primary. In otherembodiments, the cancer comprises Stomach (Gastric) Cancer. In otherembodiments, the cancer comprises Supratentorial PrimitiveNeuroectodermal Tumors. In other embodiments, the cancer comprisesCutaneous T-Cell Lymphoma, e.g., Mycosis Fungoides and Sezary Syndrome.In other embodiments, the cancer comprises Testicular Cancer. In otherembodiments, the cancer comprises Throat Cancer. In other embodiments,the cancer comprises Thymoma or Thymic Carcinoma. In other embodiments,the cancer comprises Thyroid Cancer. In other embodiments, the cancercomprises Transitional Cell Cancer of the Renal Pelvis and Ureter. Inother embodiments, the cancer comprises Gestational Trophoblastic Tumor.In other embodiments, the cancer comprises a Carcinoma of UnknownPrimary Site. In other embodiments, the cancer comprises an UnusualCancer of Childhood. In other embodiments, the cancer comprises Ureterand Renal Pelvis Transitional Cell Cancer. In other embodiments, thecancer comprises Urethral Cancer. In other embodiments, the cancercomprises Endometrial Uterine Cancer. In other embodiments, the cancercomprises Uterine Sarcoma. In other embodiments, the cancer comprisesVaginal Cancer. In other embodiments, the cancer comprises VisualPathway and Hypothalamic Glioma. In other embodiments, the cancercomprises Vulvar Cancer. In other embodiments, the cancer comprisesWaldenstrim Macroglobulinemia. In other embodiments, the cancercomprises Wilms Tumor. In other embodiments, the cancer comprisesWomen's Cancers.

Cardiovascular disease may be included in other applications of theinvention. Examples of cardiovascular disease include, but are notlimited to, congestive heart failure, high blood pressure, arrhythmias,atherosclerosis, cholesterol, Wolff-Parkinson-White Syndrome, long QTsyndrome, angina pectoris, tachycardia, bradycardia, atrialfibrillation, ventricular fibrillation, congestive heart failure,myocardial ischemia, myocardial infarction, cardiac tamponade,myocarditis, pericarditis, arrhythmogenic right ventricular dysplasia,hypertrophic cardiomyopathy, Williams syndrome, heart valve diseases,endocarditis, bacterial, pulmonary atresia, aortic valve stenosis,Raynaud's disease, cholesterol embolism, Wallenberg syndrome,Hippel-Lindau disease, and telangiectasis.

Inflammatory disease and autoimmune disease may be included in otherembodiments of the invention. Examples of inflammatory disease andautoimmune disease include, but are not limited to, rheumatoidarthritis, non-specific arthritis, inflammatory disease of the larynx,inflammatory bowel disorder, psoriasis, hypothyroidism (e.g., Hashimotothyroidism), colitis, Type 1 diabetes, pelvic inflammatory disease,inflammatory disease of the central nervous system, temporal arteritis,polymyalgia rheumatica, ankylosing spondylitis, polyarteritis nodosa,Reiter's syndrome, scleroderma, systemis lupus and erythematosus.

The methods and compositions of the invention can also providelaboratory information about markers of infectious disease includingmarkers of Adenovirus, Bordella pertussis, Chlamydia pneumoiea,Chlamydia trachomatis, Cholera Toxin, Cholera Toxin β, Campylobacterjejuni, Cytomegalovirus, Diptheria Toxin, Epstein-Barr NA, Epstein-BarrEA, Epstein-Barr VCA, Helicobacter Pylori, Hepatitis B virus (HBV) Core,Hepatitis B virus (HBV) Envelope, Hepatitis B virus (HBV) Surface (Ay),Hepatitis C virus (HCV) Core, Hepatitis C virus (HCV) NS3, Hepatitis Cvirus (HCV) NS4, Hepatitis C virus (HCV) NS5, Hepatitis A, Hepatitis D,Hepatitis E virus (HEV) orf2 3 KD, Hepatitis E virus (HEV) orf2 6 KD,Hepatitis E virus (HEV) orf3 3 KD, Human immunodeficiency virus (HIV)-1p24, Human immunodeficiency virus (HIV)-1 gp41, Human immunodeficiencyvirus (HIV)-1 gp120, Human papilloma virus (HPV), Herpes simplex virusHSV-1/2, Herpes simplex virus HSV-1 gD, Herpes simplex virus HSV-2 gG,Human T-cell leukemia virus (HTLV)-1/2, Influenza A, Influenza A H3N₂,Influenza B, Leishmania donovani, Lyme disease, Mumps, M. pneumoniae, M.tuberculosis, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, PolioVirus, Respiratory syncytial virus (RSV), Rubella, Rubeola, StreptolysinO, Tetanus Toxin, T. pallidum 15 kd, T. pallidum p47, T. cruzi,Toxoplasma, and Varicella Zoster.

III. LABELS

In some embodiments, the invention provides methods and compositionsthat include labels for the highly sensitive detection and quantitationof molecules, e.g., markers.

One skilled in the art will recognize that many strategies can be usedfor labeling target molecules to enable their detection ordiscrimination in a mixture of particles. The labels may be attached byany known means, including methods that utilize non-specific or specificinteractions of label and target. Labels may provide a detectable signalor affect the mobility of the particle in an electric field. Inaddition, labeling can be accomplished directly or through bindingpartners.

In some embodiments, the label comprises a binding partner to themolecule of interest, where the binding partner is attached to afluorescent moiety. The compositions and methods of the invention mayutilize highly fluorescent moieties, e.g., a moiety capable of emittingat least about 200 photons when simulated by a laser emitting light atthe excitation wavelength of the moiety, wherein the laser is focused ona spot not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules. Moieties suitable for thecompositions and methods of the invention are described in more detailbelow.

In some embodiments, the invention provides a label for detecting abiological molecule comprising a binding partner for the biologicalmolecule that is attached to a fluorescent moiety, wherein thefluorescent moiety is capable of emitting at least about 200 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, wherein the laser is focused on a spot not less than about 5microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the moiety comprises a plurality offluorescent entities, e.g., about 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to8, 2 to 9, 2 to 10, or about 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or3 to 10 fluorescent entities. In some embodiments, the moiety comprisesabout 2 to 4 fluorescent entities. In some embodiments, the biologicalmolecule is a protein or a small molecule. In some embodiments, thebiological molecule is a protein. The fluorescent entities can befluorescent dye molecules. In some embodiments, the fluorescent dyemolecules comprise at least one substituted indolium ring system inwhich the substituent on the 3-carbon of the indolium ring contains achemically reactive group or a conjugated substance. In someembodiments, the dye molecules are Alexa Fluor molecules selected fromthe group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor647, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the dyemolecules are Alexa Fluor molecules selected from the group consistingof Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700.In some embodiments, the dye molecules are Alexa Fluor 647 dyemolecules. In some embodiments, the dye molecules comprise a first typeand a second type of dye molecules, e.g., two different Alexa Fluormolecules, e.g., where the first type and second type of dye moleculeshave different emission spectra. The ratio of the number of first typeto second type of dye molecule can be, e.g., 4 to 1, 3 to 1, 2 to 1, 1to 1, 1 to 2, 1 to 3 or 1 to 4. The binding partner can be, e.g., anantibody.

In some embodiments, the invention provides a label for the detection ofa marker, wherein the label comprises a binding partner for the markerand a fluorescent moiety, wherein the fluorescent moiety is capable ofemitting at least about 200 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, wherein the laser isfocused on a spot not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the fluorescent moiety comprises a fluorescent molecule. In someembodiments, the fluorescent moiety comprises a plurality of fluorescentmolecules, e.g., about 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8,or 3 to 6 fluorescent molecules. In some embodiments, the labelcomprises about 2 to 4 fluorescent molecules. In some embodiments, thefluorescent dye molecules comprise at least one substituted indoliumring system in which the substituent on the 3-carbon of the indoliumring contains a chemically reactive group or a conjugated substance. Insome embodiments, the fluorescent molecules are selected from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, AlexaFluor 680 or Alexa Fluor 700. In some embodiments, the fluorescentmolecules are selected from the group consisting of Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700. In someembodiments, the fluorescent molecules are Alexa Fluor 647 molecules. Insome embodiments, the binding partner comprises an antibody. In someembodiments, the antibody is a monoclonal antibody. In otherembodiments, the antibody is a polyclonal antibody.

The antibody may be specific to any suitable marker. In someembodiments, the antibody is specific to a marker that is selected fromthe group consisting of cytokines, growth factors, oncology markers,markers of inflammation, endocrine markers, autoimmune markers, thyroidmarkers, cardiovascular markers, markers of diabetes, markers ofinfectious disease, neurological markers, respiratory markers,gastrointestinal markers, musculoskeletal markers, dermatologicaldisorders, and metabolic markers.

In some embodiments, the antibody is specific to a marker that is acytokine. In some embodiments, the cytokine is selected from the groupconsisting of BDNF, CREB pS133, CREB Total, DR-5, EGF, ENA-78, Eotaxin,Fatty Acid Binding Protein, FGF-basic, granulocyte colony-stimulatingfactor (G-CSF), GCP-2, Granulocyte-macrophage Colony-stimulating FactorGM-CSF (GM-CSF), growth-related oncogene-keratinocytes (GRO-KC), HGF,ICAM-1, IFN-alpha, IFN-gamma, interleukins such as IL-10, IL-11, IL-12,IL-12 p40, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-16, IL-17, IL-18,IL-1alpha, IL-1beta, IL-1ra, IL-1ra/IL-1F3, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, interferon-inducible protein (10 IP-10),JE/MCP-1, keratinocytes (KC), KC/GROa, LIF, Lymphotacin, M-CSF, monocytechemoattractant protein-1 (MCP-1), MCP-1(MCAF), MCP-3, MCP-5, MDC, MIG,macrophage inflammatory (MIP-1 alpha), MIP-1 beta, MIP-1 gamma, MIP-2,MIP-3 beta, OSM, PDGF-BB, regulated upon activation, normal T cell,expressed and secreted (RANTES), Rb (pT821), Rb (total), Rb pSpT249/252,Tau (pS214), Tau (pS396), Tau (total), Tissue Factor, tumor necrosisfactor-alpha (TNF-alpha), TNF-beta, TNF-R1, TNF-RII, VCAM-1, and VEGF.

In some embodiments, the cytokine is selected from the group consistingof IL-12p70, IL-10, IL-1 alpha, IL-3, IL-12 p40, IL-1ra, IL-12, IL-6,IL-4, IL-18, IL-10, IL-5, Eotaxin, IL-16, MIG, IL-8, IL-17, IL-7, IL-15,IL-13, IL-2R (soluble), IL-2, LIF/HILDA, IL-1 beta, Fas/CD95/Apo-1 andMCP-1.

In some embodiments, the antibody is specific to a marker that is agrowth factor. In some embodiments, the antibody is specific to a markerthat is a growth factor that is TGF-beta. In some embodiments, thegrowth factor is GF Ligands such as Amphiregulin, LRIG3, Betacellulin,Neuregulin-1/NRG1, EGF, Neuregulin-3/NRG3, Epigen, TGF-alpha,Epiregulin, TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, LRIG1; EGF R/ErbBReceptor Family such as EGF R, ErbB3, ErbB2, ErbB4; FGF Family such asFGF Ligands, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16,FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9,FGF-22, FGF-10, FGF-23, FGF-11, KGF/FGF-7, FGF Receptors FGF R1-4, FGFR3, FGF R1, FGF R4, FGF R2, FGF R5, FGF Regulators FGF-BP; the HedgehogFamily Desert Hedgehog, Sonic Hedgehog, Indian Hedgehog; HedgehogRelated Molecules & Regulators BOC, GLI-3, CDO, GSK-3 alpha/beta, DISP1,GSK-3 alpha, Gas1, GSK-3 beta, GLI-1, Hip, GLI-2; the IGF Family IGFLigands IGF-I, IGF-II, IGF-I Receptor (CD221)IGF-I R, and IGF BindingProtein (IGFBP) Family ALS, IGFBP-5, CTGF/CCN2, IGFBP-6, Cyr61/CCN1,IGFBP-L1, Endocan, IGFBP-rp1/IGFBP-7, IGFBP-1, IGFBP-rP10, IGFBP-2,NOV/CCN3, IGFBP-3, WISP-1/CCN4, IGFBP-4; Receptor Tyrosine Kinases Ax1,FGF R4, C1q R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF-I R,EGF, R IGF-II R, Eph, INSRR, EphA1, Insulin R/CD220, EphA2, M-CSF R,EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R alpha, EphA7,PDGF R beta, EphA8, Ret, EphB1, RTK-like Orphan Receptor 1/ROR1, EphB2,RTK-like Orphan Receptor 2/ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1,EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF, R1-4 VEGF R,FGF R1, VEGF R1/Flt-1, FGF R2, VEGF R2/KDR/Flk-1, FGF R3, VEGF R3/Flt-4;Proteoglycans & Regulators Proteoglycans Aggrecan, Mimecan, Agrin,NG2/MCSP, Biglycan, Osteoadherin, Decorin, Podocan, DSPG3,delta-Sarcoglycan, Endocan, Syndecan-1/CD138, Endoglycan, Syndecan-2,Endorepellin/Perlecan, Syndecan-3, Glypican 2, Syndecan-4, Glypican 3,Testican 1/SPOCK1, Glypican 5, Testican 2/SPOCK2, Glypican 6, Testican3/SPOCK3, Lumican, Versican, Proteoglycan Regulators, ArylsulfataseA/ARSA, Glucosamine (N-acetyl)-6-Sulfatase/GNS, Exostosin-like 2/EXTL2,HS6ST2, Exostosin-like 3/EXTL3, Iduronate 2-Sulfatase/IDS, GalNAc4S-6ST;SCF, Flt-3 Ligand & M-CSF Flt-3, M-CSF R, Flt-3 Ligand, SCF, M-CSF, SCFR/c-kit; TGF-beta Superfamily (same as listed for inflammatory markers);VEGF/PDGF Family Neuropilin-1, P1GF, Neuropilin-2, P1GF-2, PDGF, VEGF,PDGF R alpha, VEGF-B, PDGF R beta, VEGF-C, PDGF-A, VEGF-D, PDGF-AB, VEGFR, PDGF-B, VEGF R1/Flt-1, PDGF-C, VEGF R2/KDR/Flk-1, PDGF-D, VEGFR3/Flt-4; Wnt-related Molecules Dickkopf Proteins & Wnt InhibitorsDkk-1, Dkk-4, Dkk-2, Soggy-1, Dkk-3, WIF-1 Frizzled & Related ProteinsFrizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1,Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7,MFRP Wnt Ligands Wnt-1, Wnt-8a, Wnt-2b, Wnt-8b, Wnt-3a, Wnt-9a, Wnt-4,Wnt-9b, Wnt-5a, Wnt-10a, Wnt-5b, Wnt-10b, Wnt-7a, Wnt-11, Wnt-7b; OtherWnt-related Molecules APC, Kremen-2, Axin-1, LRP-1, beta-Catenin, LRP-6,Dishevelled-1, Norrin, Dishevelled-3, PKC beta 1, Glypican 3, Pygopus-1,Glypican 5, Pygopus-2, GSK-3 alpha/beta, R-Spondin 1, GSK-3 alpha,R-Spondin 2, GSK-3 beta, R-Spondin 3, ICAT, RTK-like Orphan Receptor1/ROR1, Kremen-1, RTK-like Orphan Receptor 2/ROR, and Other GrowthFactors CTGF/CCN2, beta-NGF, Cyr61/CCN1, Norrin, DANCE, NOV/CCN3,EG-VEGF/PK1, Osteocrin, Hepassocin, PD-ECGF, HGF, Progranulin, LECT2,Thrombopoietin, LEDGF, or WISP-1/CCN4.

In some embodiments, the antibody is specific to a marker that is amarker for cancer (oncology marker). In some embodiments, the antibodyis specific to a marker that is a marker for cancer that is EGF. In someembodiments, the antibody is specific to a marker that is a marker forcancer that is TNF-alpha. In some embodiments, the antibody is specificto a marker that is a marker for cancer that is PSA. In someembodiments, the antibody is specific to a marker that is a marker forcancer that is VEGF. In some embodiments, the antibody is specific to amarker that is a marker for cancer that is TGF-beta. In someembodiments, the antibody is specific to a marker that is a marker forcancer that is FGFb. In some embodiments, the antibody is specific to amarker that is a marker for cancer that is TRAIL. In some embodiments,the antibody is specific to a marker that is a marker for cancer that isTNF-RI (p55).

In further embodiments, the antibody is specific to a marker for cancerthat is alpha-Fetoprotein. In some embodiments, the antibody is specificto a marker for cancer that is ER beta/NR3A2. In some embodiments, theantibody is specific to a marker for cancer that is ErbB2. In someembodiments, the antibody is specific to a marker for cancer that isKallikrein 3/PSA. In some embodiments, the antibody is specific to amarker for cancer that is ER alpha/NR3A1. In some embodiments, theantibody is specific to a marker for cancer that is ProgesteroneR/NR3C3. In some embodiments, the antibody is specific to a marker forcancer that is A33. In some embodiments, the antibody is specific to amarker for cancer that is MIA. In some embodiments, the antibody isspecific to a marker for cancer that is Aurora A. In some embodiments,the antibody is specific to a marker for cancer that is MMP-2. In someembodiments, the antibody is specific to a marker for cancer that isBcl-2. In some embodiments, the antibody is specific to a marker forcancer that is MMP-3. In some embodiments, the antibody is specific to amarker for cancer that is Cadherin-13. In some embodiments, the antibodyis specific to a marker for cancer that is MMP-9. In some embodiments,the antibody is specific to a marker for cancer that is E-Cadherin. Insome embodiments, the antibody is specific to a marker for cancer thatis NEK2. In some embodiments, the antibody is specific to a marker forcancer that is Carbonic Anhydrase IX. In some embodiments, the antibodyis specific to a marker for cancer that is Nestin. In some embodiments,the antibody is specific to a marker for cancer that is beta-Catenin. Insome embodiments, the antibody is specific to a marker for cancer thatis NG2/MCSP. In some embodiments, the antibody is specific to a markerfor cancer that is Cathepsin D. In some embodiments, the antibody isspecific to a marker for cancer that is Osteopontin. In someembodiments, the antibody is specific to a marker for cancer that isCD44. In some embodiments, the antibody is specific to a marker forcancer that is p21/CIP1/CDKN1A. In some embodiments, the antibody isspecific to a marker for cancer that is CEACAM-6. In some embodiments,the antibody is specific to a marker for cancer that is p27/Kip1. Insome embodiments, the antibody is specific to a marker for cancer thatis Cornulin. In some embodiments, the antibody is specific to a markerfor cancer that is p53. In some embodiments, the antibody is specific toa marker for cancer that is DPPA4. In some embodiments, the antibody isspecific to a marker for cancer that is Prolactin. In some embodiments,the antibody is specific to a marker for cancer that is ECM-1. In someembodiments, the antibody is specific to a marker for cancer that isPSP94. In some embodiments, the antibody is specific to a marker forcancer that is EGF. In some embodiments, the antibody is specific to amarker for cancer that is S100B. In some embodiments, the antibody isspecific to a marker for cancer that is EGF R. In some embodiments, theantibody is specific to a marker for cancer that is S100P. In someembodiments, the antibody is specific to a marker for cancer that isEMMPRIN/CD147. In some embodiments, the antibody is specific to a markerfor cancer that is SCF R/c-kit. In some embodiments, the antibody isspecific to a marker for cancer that is Fibroblast Activation Proteinalpha/FAP. In some embodiments, the antibody is specific to a marker forcancer that is Serpin E1/PAI-1. In some embodiments, the antibody isspecific to a marker for cancer that is FGF acidic. In some embodiments,the antibody is specific to a marker for cancer that is Serum AmyloidA4. In some embodiments, the antibody is specific to a marker for cancerthat is FGF basic. In some embodiments, the antibody is specific to amarker for cancer that is Survivin. In some embodiments, the antibody isspecific to a marker for cancer that is Galectin-3. In some embodiments,the antibody is specific to a marker for cancer that is TEM8. In someembodiments, the antibody is specific to a marker for cancer that isGlypican 3. In some embodiments, the antibody is specific to a markerfor cancer that is TIMP-1. In some embodiments, the antibody is specificto a marker for cancer that is HIN-1/Secretoglobulin 3A1. In someembodiments, the antibody is specific to a marker for cancer that isTIMP-2. In some embodiments, the antibody is specific to a marker forcancer that is IGF-I. In some embodiments, the antibody is specific to amarker for cancer that is TIMP-3. In some embodiments, the antibody isspecific to a marker for cancer that is IGFBP-3. In some embodiments,the antibody is specific to a marker for cancer that is TIMP-4. In someembodiments, the antibody is specific to a marker for cancer that isIL-6. In some embodiments, the antibody is specific to a marker forcancer that is TNF-alpha/TNFSF1A. In some embodiments, the antibody isspecific to a marker for cancer that is Kallikrein 6/Neurosin. In someembodiments, the antibody is specific to a marker for cancer that isTRAF-4. In some embodiments, the antibody is specific to a marker forcancer that is M-CSF. In some embodiments, the antibody is specific to amarker for cancer that is uPA. In some embodiments, the antibody isspecific to a marker for cancer that is Matriptase/ST14. In someembodiments, the antibody is specific to a marker for cancer that isuPAR. In some embodiments, the antibody is specific to a marker forcancer that is Mesothelin. In some embodiments, the antibody is specificto a marker for cancer that is VCAM-1. In some embodiments, the antibodyis specific to a marker for cancer that is Methionine Aminopeptidase. Insome embodiments, the antibody is specific to a marker for cancer thatis VEGF. In some embodiments, the antibody is specific to a marker forcancer that is Methionine Aminopeptidase 2.

In some embodiments, the antibody is specific to a marker that is amarker for inflammation. In some embodiments, the antibody is specificto a marker that is a marker for inflammation that is ICAM-1. In someembodiments, the antibody is specific to a marker that is a marker forinflammation that is RANTES. In some embodiments, the antibody isspecific to a marker that is a marker for inflammation that is MIP-2. Insome embodiments, the antibody is specific to a marker that is a markerfor inflammation that is MIP-1 beta. In some embodiments, the antibodyis specific to a marker that is a marker for inflammation that is MIP-1alpha. In some embodiments, the antibody is specific to a marker that isa marker for inflammation that is MMP-3.

In some embodiments, the antibody is specific to a marker that is amarker for endocrine function. In some embodiments, the antibody isspecific to a marker that is a marker for endocrine function that is 17beta-estradiol (E2). In some embodiments, the antibody is specific to amarker that is a marker for endocrine function that is DHEA. In someembodiments, the antibody is specific to a marker that is a marker forendocrine function that is ACTH. In some embodiments, the antibody isspecific to a marker that is a marker for endocrine function that isgastrin. In some embodiments, the antibody is specific to a marker thatis a marker for endocrine function that is growth hormone.

In some embodiments, the antibody is specific to a marker that is amarker for autoimmune disease. In some embodiments, the antibody isspecific to a marker that is a marker for autoimmune disease that isGM-CSF. In some embodiments, the antibody is specific to a marker thatis a marker for autoimmune disease that is C-reactive protein (CRP). Insome embodiments, the antibody is specific to a marker that is a markerfor autoimmune disease that is G-CSF.

In some embodiments, the antibody is specific to a marker for thyroidfunction. In some embodiments, the antibody is specific to a marker forthyroid function that is cyclic AMP. In some embodiments, the antibodyis specific to a marker for thyroid function. In some embodiments, theantibody is specific to a marker for thyroid function that iscalcitonin. In some embodiments, the antibody is specific to a markerfor thyroid function. In some embodiments, the antibody is specific to amarker for thyroid function that is parathyroid hormone.

In some embodiments, the antibody is specific to a marker forcardiovascular function. In some embodiments, the antibody is specificto a marker for cardiovascular function that is B-natriuretic peptide.In some embodiments, the antibody is specific to a marker forcardiovascular function that is NT-proBNP. In some embodiments, theantibody is specific to a marker for cardiovascular function that isC-reactive protein, HS. In some embodiments, the antibody is specific toa marker for cardiovascular function that is beta-thromboglobulin. Insome embodiments, the antibody is specific to a marker forcardiovascular function that is a cardiac troponin. In some embodiments,the antibody is specific to a marker for cardiovascular function that iscardiac troponin I. In some embodiments, the antibody is specific to amarker for cardiovascular function that is cardiac troponin T.

In some embodiments, the antibody is specific to a marker for diabetes.In some embodiments, the antibody is specific to a marker for diabetesthat is C-peptide. In some embodiments, the antibody is specific to amarker for diabetes that is leptin.

In some embodiments, the antibody is specific to a marker for infectiousdisease. In some embodiments, the antibody is specific to a marker forinfectious disease that is IFN gamma. In some embodiments, the antibodyis specific to a marker for infectious disease that is IFN alpha. Insome embodiments, the antibody is specific to a marker for infectiousdisease that is TREM-1.

In some embodiments, the antibody is specific to a marker formetabolism. In some embodiments, the antibody is specific to a markerfor metabolism that is bio-intact PTH (1-84). In some embodiments, theantibody is specific to a marker for metabolism that is PTH.

In some embodiments, the antibody is specific to a marker that is IL-1beta. In some embodiments, the antibody is specific to a marker that isTNF-alpha. In some embodiments, the antibody is specific to a markerthat is IL-6. In some embodiments, the antibody is specific to a markerthat is TnI (cardiac troponin I). In some embodiments, the antibody isspecific to a marker that is IL-8.

In some embodiments, the antibody is specific to a marker that is Abeta40. In some embodiments, the antibody is specific to a marker that isAbeta 42. In some embodiments, the antibody is specific to a marker thatis cAMP. In some embodiments, the antibody is specific to a marker thatis FAS Ligand. In some embodiments, the antibody is specific to a markerthat is FGF-basic. In some embodiments, the antibody is specific to amarker that is GM-CSF. In some embodiments, the antibody is specific toa marker that is IFN-alpha. In some embodiments, the antibody isspecific to a marker that is IFN-gamma. In some embodiments, theantibody is specific to a marker that is IL-1a. In some embodiments, theantibody is specific to a marker that is IL-2. In some embodiments, theantibody is specific to a marker that is IL-4. In some embodiments, theantibody is specific to a marker that is IL-5. In some embodiments, theantibody is specific to a marker that is IL-7. In some embodiments, theantibody is specific to a marker that is IL-12. In some embodiments, theantibody is specific to a marker that is In some embodiments, theantibody is specific to a marker that is IL-13. In some embodiments, theantibody is specific to a marker that is IL-17. In some embodiments, theantibody is specific to a marker that is MCP-1. In some embodiments, theantibody is specific to a marker that is MIP-1a. In some embodiments,the antibody is specific to a marker that is RANTES. In someembodiments, the antibody is specific to a marker that is VEGF.

In some embodiments, the antibody is specific to a marker that is ACE.In some embodiments, the antibody is specific to a marker that isactivin A. In some embodiments, the antibody is specific to a markerthat is adiponectin. In some embodiments, the antibody is specific to amarker that is adipsin. In some embodiments, the antibody is specific toa marker that is AgRP. In some embodiments, the antibody is specific toa marker that is AKT1. In some embodiments, the antibody is specific toa marker that is albumin. In some embodiments, the antibody is specificto a marker that is betacellulin. In some embodiments, the antibody isspecific to a marker that is bombesin. In some embodiments, the antibodyis specific to a marker that is CD14. In some embodiments, the antibodyis specific to a marker that is CD-26. In some embodiments, the antibodyis specific to a marker that is CD-38. In some embodiments, the antibodyis specific to a marker that is CD-40L. In some embodiments, theantibody is specific to a marker that is CD-40s. In some embodiments,the antibody is specific to a marker that is CDK5. In some embodiments,the antibody is specific to a marker that is Complement C3. In someembodiments, the antibody is specific to a marker that is Complement C4.In some embodiments, the antibody is specific to a marker that isC-peptide. In some embodiments, the antibody is specific to a markerthat is CRP. In some embodiments, the antibody is specific to a markerthat is EGF. In some embodiments, the antibody is specific to a markerthat is E-selectin. In some embodiments, the antibody is specific to amarker that is FAS. In some embodiments, the antibody is specific to amarker that is FASLG. In some embodiments, the antibody is specific to amarker that is Fetuin A. In some embodiments, the antibody is specificto a marker that is fibrinogen. In some embodiments, the antibody isspecific to a marker that is ghrelin. In some embodiments, the antibodyis specific to a marker that is glucagon. In some embodiments, theantibody is specific to a marker that is growth hormone. In someembodiments, the antibody is specific to a marker that is haptoglobulin.In some embodiments, the antibody is specific to a marker that ishepatocyte growth factor. In some embodiments, the antibody is specificto a marker that is HGF. In some embodiments, the antibody is specificto a marker that is ICAM1. In some embodiments, the antibody is specificto a marker that is IFNG. In some embodiments, the antibody is specificto a marker that is IGF1. In some embodiments, the antibody is specificto a marker that is IL-1RA. In some embodiments, the antibody isspecific to a marker that is Il-6sr. In some embodiments, the antibodyis specific to a marker that is IL-8. In some embodiments, the antibodyis specific to a marker that is IL-10. In some embodiments, the antibodyis specific to a marker that is IL-18. In some embodiments, the antibodyis specific to a marker that is ILGFBP1. In some embodiments, theantibody is specific to a marker that is ILGFBP3. In some embodiments,the antibody is specific to a marker that is insulin-like growthfactor 1. In some embodiments, the antibody is specific to a marker thatis LEP. In some embodiments, the antibody is specific to a marker thatis M-CSF. In some embodiments, the antibody is specific to a marker thatis MMP2. In some embodiments, the antibody is specific to a marker thatis MMP9. In some embodiments, the antibody is specific to a marker thatis NGF. In some embodiments, the antibody is specific to a marker thatis PAI-1. In some embodiments, the antibody is specific to a marker thatis RAGE. In some embodiments, the antibody is specific to a marker thatis RSP4. In some embodiments, the antibody is specific to a marker thatis resistin. In some embodiments, the antibody is specific to a markerthat is sex hormone binding globulin. In some embodiments, the antibodyis specific to a marker that is SOCX3. In some embodiments, the antibodyis specific to a marker that is TGF beta. In some embodiments, theantibody is specific to a marker that is thromboplastin. In someembodiments, the antibody is specific to a marker that is TNF R1. Insome embodiments, the antibody is specific to a marker that is VCAM-1.In some embodiments, the antibody is specific to a marker that is VWF.In some embodiments, the antibody is specific to a marker that is TSH.In some embodiments, the antibody is specific to a marker that isEPITOME.

In some embodiments, the antibody is specific to a marker that iscardiac troponin I. In some embodiments, the antibody is specific to amarker that is TREM-1. In some embodiments, the antibody is specific toa marker that is IL-6. In some embodiments, the antibody is specific toa marker that is IL-8. In some embodiments, the antibody is specific toa marker that is Leukotriene T4. In some embodiments, the antibody isspecific to a marker that is Akt1. In some embodiments, the antibody isspecific to a marker that is TGF-beta. In some embodiments, the antibodyis specific to a marker that is Fas ligand.

In some embodiments, the fluorescent moiety comprises a fluorescentmolecule. In some embodiments, the fluorescent moiety comprises aplurality of fluorescent molecules, e.g., about 2 to 10, 2 to 8, 2 to 6,2 to 4, 3 to 10, 3 to 8, or 3 to 6 fluorescent molecules. In someembodiments, the label comprises about 2 to 4 fluorescent molecules. Insome embodiments, the fluorescent molecule comprises a molecule thatcomprises at least one substituted indolium ring system in which thesubstituent on the 3-carbon of the indolium ring contains a chemicallyreactive group or a conjugated substance group. In some embodiments, thefluorescent molecules are selected from the group consisting of AlexaFluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or AlexaFluor 700. In some embodiments, the fluorescent molecules are selectedfrom the group consisting of Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 680 or Alexa Fluor 700. In some embodiments, the fluorescentmolecules are Alexa Fluor 647 molecules.

A. Binding partners

Any suitable binding partner with the requisite specificity for the formof molecule, e.g., a marker, to be detected may be used. If themolecule, e.g., a marker, has several different forms, variousspecificities of binding partners are possible. Suitable bindingpartners are known in the art and include antibodies, aptamers, lectins,and receptors. A useful and versatile type of binding partner is anantibody.

1. Antibodies

In some embodiments, the binding partner is an antibody specific for amolecule to be detected. The term “antibody,” as used herein, is a broadterm and is used in its ordinary sense, including, without limitation,to refer to naturally occurring antibodies as well as non-naturallyoccurring antibodies, including, for example, single chain antibodies,chimeric, bifunctional and humanized antibodies, as well asantigen-binding fragments thereof. It will be appreciated that thechoice of epitope or region of the molecule to which the antibody israised will determine its specificity, e.g., for various forms of themolecule, if present, or for total (e.g., all, or substantially all ofthe molecule).

Methods for producing antibodies are well-established. One skilled inthe art will recognize that many procedures are available for theproduction of antibodies, for example, as described in Antibodies, ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art willalso appreciate that binding fragments or Fab fragments which mimicantibodies can also be prepared from genetic information by variousprocedures (Antibody Engineering: A Practical Approach (Borrebaeck, C.,ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920(1992)). Monoclonal and polyclonal antibodies to molecules, e.g.,proteins, and markers also commercially available (R and D Systems,Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abcam Inc.,Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA;Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049USA; BiosPacific, Emeryville, Calif.).

In some embodiments, the antibody is a polyclonal antibody. In otherembodiments, the antibody is a monoclonal antibody.

Capture binding partners and detection binding partner pairs, e.g.,capture and detection antibody pairs, may be used in embodiments of theinvention. Thus, in some embodiments, a heterogeneous assay protocol isused in which, typically, two binding partners, e.g., two antibodies,are used. One binding partner is a capture partner, usually immobilizedon a solid support, and the other binding partner is a detection bindingpartner, typically with a detectable label attached. Such antibody pairsare available from the sources described above, e.g., BiosPacific,Emeryville, Calif. Antibody pairs can also be designed and prepared bymethods well-known in the art. Compositions of the invention includeantibody pairs wherein one member of the antibody pair is a label asdescribed herein, and the other member is a capture antibody.

In some embodiments it is useful to use an antibody that cross-reactswith a variety of species, either as a capture antibody, a detectionantibody, or both. Such embodiments include the measurement of drugtoxicity by determining, e.g., release of cardiac troponin into theblood as a marker of cardiac damage. A cross-reacting antibody allowsstudies of toxicity to be done in one species, e.g., a non-humanspecies, and direct transfer of the results to studies or clinicalobservations of another species, e.g., humans, using the same antibodyor antibody pair in the reagents of the assays, thus decreasingvariability between assays. Thus, in some embodiments, one or more ofthe antibodies for use as a binding partner to the marker, e.g., cardiactroponin, such as cardiac troponin I, may be a cross-reacting antibody.In some embodiments, the antibody cross-reacts with the marker, e.g.,cardiac troponin, from at least two species selected from the groupconsisting of human, monkey, dog, and mouse. In some embodiments theantibody cross-reacts with the marker e.g., cardiac troponin, from allof the group consisting of human, monkey, dog, and mouse.

B. Fluorescent Moieties

In some embodiments of labels used in the invention, the bindingpartner, e.g., antibody, is attached to a fluorescent moiety. Thefluorescence of the moiety will be sufficient to allow detection in asingle molecule detector, such as the single molecule detectorsdescribed herein.

A “fluorescent moiety,” as that term is used herein, includes one ormore fluorescent entities whose total fluorescence is such that themoiety may be detected in the single molecule detectors describedherein. Thus, a fluorescent moiety may comprise a single entity (e.g., aQuantum Dot or fluorescent molecule) or a plurality of entities (e.g., aplurality of fluorescent molecules). It will be appreciated that when“moiety,” as that term is used herein, refers to a group of fluorescententities, e.g., a plurality of fluorescent dye molecules, eachindividual entity may be attached to the binding partner separately orthe entities may be attached together, as long as the entities as agroup provide sufficient fluorescence to be detected.

Typically, the fluorescence of the moiety involves a combination ofquantum efficiency and lack of photobleaching sufficient that the moietyis detectable above background levels in a single molecule detector,with the consistency necessary for the desired limit of detection,accuracy, and precision of the assay. For example, in some embodiments,the fluorescence of the fluorescent moiety is such that it allowsdetection and/or quantitation of a molecule, e.g., a marker, at a limitof detection of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001,0.00001, or 0.000001 pg/ml and with a coefficient of variation of lessthan about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% orless, e.g., about 10% or less, in the instruments described herein. Insome embodiments, the fluorescence of the fluorescent moiety is suchthat it allows detection and/or quantitation of a molecule, e.g., amarker, at a limit of detection of less than about 5, 1, 0.5, 0.1, 0.05,0.01, 0.005, 0.001 pg/ml and with a coefficient of variation of lessthan about 10%, in the instruments described herein.

“Limit of detection,” or LoD, as those terms are used herein, includesthe lowest concentration at which one can identify a sample ascontaining a molecule of the substance of interest, e.g., the firstnon-zero value. It can be defined by the variability of zeros and theslope of the standard curve. For example, the limit of detection of anassay may be determined by running a standard curve, determining thestandard curve zero value, and adding 2 standard deviations to thatvalue. A concentration of the substance of interest that produces asignal equal to this value is the “lower limit of detection”concentration.

Furthermore, the moiety has properties that are consistent with its usein the assay of choice. In some embodiments, the assay is animmunoassay, where the fluorescent moiety is attached to an antibody;the moiety must have properties such that it does not aggregate withother antibodies or proteins, or experiences no more aggregation than isconsistent with the required accuracy and precision of the assay. Insome embodiments, fluorescent moieties that are preferred arefluorescent moieties, e.g., dye molecules that have a combination of 1)high absorption coefficient; 2) high quantum yield; 3) highphotostability (low photobleaching); and 4) compatibility with labelingthe molecule of interest (e.g., protein) so that it may be analyzedusing the analyzers and systems of the invention (e.g., does not causeprecipitation of the protein of interest, or precipitation of a proteinto which the moiety has been attached).

Fluorescent moieties, e.g., a single fluorescent dye molecule or aplurality of fluorescent dye molecules, that are useful in someembodiments of the invention may be defined in terms of their photonemission characteristics when stimulated by EM radiation. For example,in some embodiments, the invention utilizes a fluorescent moiety, e.g.,a moiety comprising a single fluorescent dye molecule or a plurality offluorescent dye molecules, that is capable of emitting an average of atleast about 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 350, 400, 500, 600, 700, 800, 900, or 1000, photons whensimulated by a laser emitting light at the excitation wavelength of themoiety, where the laser is focused on a spot of not less than about 5microns in diameter that contains the moiety, and where the total energydirected at the spot by the laser is no more than about 3 microJoules.It will be appreciated that the total energy may be achieved by manydifferent combinations of power output of the laser and length of timeof exposure of the dye moiety. E.g., a laser of a power output of 1 mWmay be used for 3 ms, 3 mW for 1 ms, 6 mW for 0.5 ms, 12 mW for 0.25 ms,and so on.

In some embodiments, the invention utilizes a fluorescent dye moiety,e.g., a single fluorescent dye molecule or a plurality of fluorescentdye molecules, that is capable of emitting an average of at least about50 photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the invention utilizes afluorescent dye moiety, e.g., a single fluorescent dye molecule or aplurality of fluorescent dye molecules, that is capable of emitting anaverage of at least about 100 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, where the laser isfocused on a spot of not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the invention utilizes a fluorescent dye moiety, e.g., a singlefluorescent dye molecule or a plurality of fluorescent dye molecules,that is capable of emitting an average of at least about 150 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, where the laser is focused on a spot of not less than about5 microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the invention utilizes a fluorescentdye moiety, e.g., a single fluorescent dye molecule or a plurality offluorescent dye molecules, that is capable of emitting an average of atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, where the laser is focused on aspot of not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules. In some embodiments, the inventionutilizes a fluorescent dye moiety, e.g., a single fluorescent dyemolecule or a plurality of fluorescent dye molecules, that is capable ofemitting an average of at least about 300 photons when simulated by alaser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the moiety, and wherein the total energy directedat the spot by the laser is no more than about 3 microJoules. In someembodiments, the invention utilizes a fluorescent dye moiety e.g., asingle fluorescent dye molecule or a plurality of fluorescent dyemolecules, that is capable of emitting an average of at least about 500photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules.

In some embodiments, the fluorescent moiety comprises an average of atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorescent entities, e.g.,fluorescent molecules. In some embodiments, the fluorescent moietycomprises an average of no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 fluorescent entities, e.g., fluorescent molecules. In someembodiments, the fluorescent moiety comprises an average of about 1 to11, or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5,or about 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, orabout 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, orabout 2, 3, 4, 5, 6, or more than about 6 fluorescent entities. In someembodiments, the fluorescent moiety comprises an average of about 2 to 8fluorescent moieties are attached. In some embodiments, the fluorescentmoiety comprises an average of about 2 to 6 fluorescent entities. Insome embodiments, the fluorescent moiety comprises an average of about 2to 4 fluorescent entities. In some embodiments, the fluorescent moietycomprises an average of about 3 to 10 fluorescent entities. In someembodiments, the fluorescent moiety comprises an average of about 3 to 8fluorescent entities. In some embodiments, the fluorescent moietycomprises an average of about 3 to 6 fluorescent entities. By “average”it is meant that, in a given sample that is representative of a group oflabels of the invention, where the sample contains a plurality of thebinding partner-fluorescent moiety units, the molar ratio of theparticular fluorescent entity to the binding partner, as determined bystandard analytical methods, corresponds to the number or range ofnumbers specified. For example, in embodiments wherein the labelcomprises a binding partner that is an antibody and a fluorescent moietythat comprises a plurality of fluorescent dye molecules of a specificabsorbance, a spectrophotometric assay can be used in which a solutionof the label is diluted to an appropriate level and the absorbance at280 nm is taken to determine the molarity of the protein (antibody) andan absorbance at, e.g., 650 nm (for Alexa Fluor 647), is taken todetermine the molarity of the fluorescent dye molecule. The ratio of thelatter molarity to the former represents the average number offluorescent entities (dye molecules) in the fluorescent moiety attachedto each antibody.

1. Dyes

In some embodiments, the invention uses fluorescent moieties thatcomprise fluorescent dye molecules. In some embodiments, the inventionutilizes a fluorescent dye molecule that is capable of emitting anaverage of at least about 50 photons when simulated by a laser emittinglight at the excitation wavelength of the molecule, where the laser isfocused on a spot of not less than about 5 microns in diameter thatcontains the molecule, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the invention utilizes a fluorescent dye molecule that is capable ofemitting an average of at least about 75 photons when simulated by alaser emitting light at the excitation wavelength of the molecule, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the molecule, and wherein the total energydirected at the spot by the laser is no more than about 3 microJoules.In some embodiments, the invention utilizes a fluorescent dye moleculethat is capable of emitting an average of at least about 100 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe molecule, where the laser is focused on a spot of not less thanabout 5 microns in diameter that contains the molecule, and wherein thetotal energy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the invention utilizes a fluorescentdye molecule that is capable of emitting an average of at least about150 photons when simulated by a laser emitting light at the excitationwavelength of the molecule, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the molecule, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the invention utilizes afluorescent dye molecule that is capable of emitting an average of atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the molecule, where the laser is focused on aspot of not less than about 5 microns in diameter that contains themolecule, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules.

In some embodiments, the invention uses a fluorescent dye moiety, e.g.,a single fluorescent dye molecule or a plurality of fluorescent dyemolecules, that is capable of emitting an average of at least about 50photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the invention utilizes afluorescent dye moiety, e.g., a single fluorescent dye molecule or aplurality of fluorescent dye molecules, that is capable of emitting anaverage of at least about 100 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, where the laser isfocused on a spot of not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the invention utilizes a fluorescent dye moiety, e.g., a singlefluorescent dye molecule or a plurality of fluorescent dye molecules,that is capable of emitting an average of at least about 150 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, where the laser is focused on a spot of not less than about5 microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the invention utilizes a fluorescentdye moiety, e.g., a single fluorescent dye molecule or a plurality offluorescent dye molecules, that is capable of emitting an average of atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, where the laser is focused on aspot of not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules. In some embodiments, the inventionutilizes a fluorescent dye moiety, e.g., a single fluorescent dyemolecule or a plurality of fluorescent dye molecules, that is capable ofemitting an average of at least about 300 photons when simulated by alaser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the moiety, and wherein the total energy directedat the spot by the laser is no more than about 3 microJoules. In someembodiments, the invention utilizes a fluorescent dye moiety, e.g., asingle fluorescent dye molecule or a plurality of fluorescent dyemolecules, that is capable of emitting an average of at least about 500photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules.

A non-inclusive list of useful fluorescent entities for use in thefluorescent moieties of the invention is given in Table 2, below. Insome embodiments, the fluorescent dye is selected from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, AlexaFluor 700, Alexa Fluor 750, Fluorescein, B-phycoerythrin,allophycocyanin, PBXL-3, and Qdot 605. In some embodiments, thefluorescent dye is selected from the group consisting of Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 700, Alexa Fluor 750, Fluorescein,B-phycoerythrin, allophycocyanin, PBXL-3, and Qdot 605.

TABLE 2 FLUORESCENT ENTITIES Dye E Ex (nm) E (M)−1 Em (nm) MMw Bimane380 5,700 458 282.31 Dapoxyl 373 22,000 551 362.83 Dimethylaminocoumarin-4-acetic acid 375 22,000 470 344.32 Marina blue 365 19,000 460367.26 8-Anilino naphthalene-1-sulfonic acid 372 480 Cascade blue 37623,000 420 607.42 Alexa Fluor 405 402 35,000 421 1028.26 Cascade blue400 29,000 420 607.42 Cascade yellow 402 24,000 545 563.54 Pacific blue410 46,000 455 339.21 PyMPO 415 26,000 570 582.41 Alexa Fluor 430 43315,000 539 701.75 Atto-425 438 486 NBD 465 22,000 535 391.34 Alexa Fluor488 495 73,000 519 643.41 Fluorescein 494 79,000 518 376.32 Oregon Green488 496 76,000 524 509.38 Atto 495 495 522 Cy2 489 150,000 506 713.78DY-480-XL 500 40,000 630 514.60 DY-485-XL 485 20,000 560 502.59DY-490-XL 486 27,000 532 536.58 DY-500-XL 505 90,000 555 596.68DY-520-XL 520 40,000 664 514.60 Alexa Fluor 532 531 81,000 554 723.77BODIPY 530/550 534 77,000 554 513.31 6-HEX 535 98,000 556 680.07 6-JOE522 75,000 550 602.34 Rhodamine 6G 525 108,000 555 555.59 Atto-520 520542 Cy3B 558 130,000 572 658.00 Alexa Fluor 610 612 138,000 628 AlexaFluor 633 632 159,000 647 ca. 1200 Alexa Fluor 647 650 250,000 668 ca.1250 BODIPY 630/650 625 101,000 640 660.50 Cy5 649 250,000 670 791.99Alexa Fluor 660 663 110,000 690 Alexa Fluor 680 679 184,000 702 AlexaFluor 700 702 192,000 723 Alexa Fluor 750 749 240,000 782B-phycoerythrin 546, 565 2,410,000 575 240,000 R-phycoerythrin 480, 546,1,960,000 578 240,000 565 Allophycocyanin 650 700,000 660 700,000 PBXL-1545 666 PBXL-3 614 662 Atto-tec dyes Name Ex (nm) Em (nm) QY τ (ns) Atto425 436 486 0.9 3.5 Atto 495 495 522 0.45 2.4 Atto 520 520 542 0.9 3.6Atto 560 561 585 0.92 3.4 Atto 590 598 634 0.8 3.7 Atto 610 605 630 0.73.3 Atto 655 665 690 0.3 1.9 Atto 680 680 702 0.3 1.8 Dyomics FluorsMolar absorbance* molecular Label Ex (nm) [l · mol−1 · cm−1] Em (nm)weight# [g · mol−1] DY-495/5 495 70,000 520 489.47 DY-495/6 495 70,000520 489.47 DY-495X/5 495 70,000 520 525.95 DY-495X/6 495 70,000 520525.95 DY-505/5 505 85,000 530 485.49 DY-505/6 505 85,000 530 485.49DY-505X/5 505 85,000 530 523.97 DY-505X/6 505 85,000 530 523.97 DY-550553 122,000 578 667.76 DY-555 555 100.000 580 636.18 DY-610 609 81.000629 667.75 DY-615 621 200.000 641 578.73 DY-630 636 200.000 657 634.84DY-631 637 185.000 658 736.88 DY-633 637 180.000 657 751.92 DY-635 647175.000 671 658.86 DY-636 645 190.000 671 760.91 DY-650 653 170.000 674686.92 DY-651 653 160.000 678 888.96 DYQ-660 660 117,000 — 668.86DYQ-661 661 116,000 — 770.90 DY-675 674 110.000 699 706.91 DY-676 674145.000 699 807.95 DY-680 690 125.000 709 634.84 DY-681 691 125.000 708736.88 DY-700 702 96.000 723 668.86 DY-701 706 115.000 731 770.90 DY-730734 185.000 750 660.88 DY-731 736 225.000 759 762.92 DY-750 747 240.000776 712.96 DY-751 751 220.000 779 814.99 DY-776 771 147.000 801 834.98DY-780-OH 770 70.000 810 757.34 DY-780-P 770 70.000 810 957.55 DY-781783 98.000 800 762.92 DY-782 782 102.000 800 660.88 EVOblue-10 651101.440 664 389.88 EVOblue-30 652 102.000 672 447.51 Quantum Dots: Qdot525, QD 565, QD 585, QD 605, QD 655, QD 705, QD 800

Suitable dyes for use in the invention include modified carbocyaninedyes. On such modification comprises modification of an indolium ring ofthe carbocyanine dye to permit a reactive group or conjugated substanceat the number three position. The modification of the indolium ringprovides dye conjugates that are uniformly and substantially morefluorescent on proteins, nucleic acids and other biopolymers, thanconjugates labeled with structurally similar carbocyanine dyes boundthrough the nitrogen atom at the number one position. In addition tohaving more intense fluorescence emission than structurally similar dyesat virtually identical wavelengths, and decreased artifacts in theirabsorption spectra upon conjugation to biopolymers, the modifiedcarbocyanine dyes have greater photostability and higher absorbance(extinction coefficients) at the wavelengths of peak absorbance than thestructurally similar dyes. Thus, the modified carbocyanine dyes resultin greater sensitivity in assays using the modified dyes and theirconjugates. Preferred modified dyes include compounds that have at leastone substituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance. Other dye compounds include compounds thatincorporate an azabenzazolium ring moiety and at least one sulfonatemoiety. The modified carbocyanine dyes that can be used to detectindividual molecules in various embodiments of the invention aredescribed in U.S. Pat. No. 6,977,305, which is herein incorporated byreference in its entirety. Thus, in some embodiments the labels of theinvention utilize a fluorescent dye that includes a substituted indoliumring system in which the substituent on the 3-carbon of the indoliumring contains a chemically reactive group or a conjugated substancegroup.

In some embodiments, the label comprises a fluorescent moiety thatincludes one or more Alexa Fluor dyes (Molecular Probes, Eugene, Oreg.).The Alexa Fluor dyes are disclosed in U.S. Pat. Nos. 6,977,305;6,974,874; 6,130,101; and 6,974,305 which are herein incorporated byreference in their entirety. Some embodiments of the invention utilize adye chosen from the group consisting of Alexa Fluor 647, Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680,Alexa Fluor 700, and Alexa Fluor 750. Some embodiments of the inventionutilize a dye chosen from the group consisting of Alexa Fluor 488, AlexaFluor 532, Alexa Fluor 647, Alexa Fluor 700 and Alexa Fluor 750. Someembodiments of the invention utilize a dye chosen from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, AlexaFluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. Someembodiments of the invention utilize the Alexa Fluor 647 molecule, whichhas an absorption maximum between about 650 and 660 nm and an emissionmaximum between about 660 and 670 nm. The Alexa Fluor 647 dye is usedalone or in combination with other Alexa Fluor dyes.

Currently available organic fluors can be improved by rendering themless hydrophobic by adding hydrophilic groups such as polyethylene.Alternatively, currently sulfonated organic fluors such as the AlexaFluor 647 dye can be rendered less acidic by making them zwitterionic.Particles such as antibodies that are labeled with the modified fluorsare less likely to bind non-specifically to surfaces and proteins inimmunoassays, and thus enable assays that have greater sensitivity andlower backgrounds. Methods for modifying and improving the properties offluorescent dyes for the purpose of increasing the sensitivity of asystem that detects single molecules are known in the art. Preferably,the modification improves the Stokes shift while maintaining a highquantum yield.

2. Quantum dots

In some embodiments, the fluorescent label moiety that is used to detecta molecule in a sample using the analyzer systems of the invention is aquantum dot. Quantum dots (QDs), also known as semiconductornanocrystals or artificial atoms, are semiconductor crystals thatcontain anywhere between 100 to 1,000 electrons and range from 2-10 nm.Some QDs can be between 10-20 nm in diameter. QDs have high quantumyields, which makes them particularly useful for optical applications.QDs are fluorophores that fluoresce by forming excitons, which aresimilar to the excited state of traditional fluorophores, but have muchlonger lifetimes of up to 200 nanoseconds. This property provides QDswith low photobleaching. The energy level of QDs can be controlled bychanging the size and shape of the QD, and the depth of the QDs'potential. One optical feature of small excitonic QDs is coloration,which is determined by the size of the dot. The larger the dot, theredder, or more towards the red end of the spectrum the fluorescence.The smaller the dot, the bluer or more towards the blue end it is. Thebandgap energy that determines the energy and hence the color of thefluoresced light is inversely proportional to the square of the size ofthe QD. Larger QDs have more energy levels which are more closelyspaced, thus allowing the QD to absorb photons containing less energy,i.e., those closer to the red end of the spectrum. Because the emissionfrequency of a dot is dependent on the bandgap, it is possible tocontrol the output wavelength of a dot with extreme precision. In someembodiments the protein that is detected with the single moleculeanalyzer system is labeled with a QD. In some embodiments, the singlemolecule analyzer is used to detect a protein labeled with one QD andusing a filter to allow for the detection of different proteins atdifferent wavelengths.

QDs have broad excitation and narrow emission properties which, whenused with color filtering, require only a single electromagnetic sourceto resolve individual signals during multiplex analysis of multipletargets in a single sample. Thus, in some embodiments, the analyzersystem comprises one continuous wave laser and particles that are eachlabeled with one QD. Colloidally prepared QDs are free floating and canbe attached to a variety of molecules via metal coordinating functionalgroups. These groups include but are not limited to thiol, amine,nitrile, phosphine, phosphine oxide, phosphonic acid, carboxylic acidsor other ligands. By bonding appropriate molecules to the surface, thequantum dots can be dispersed or dissolved in nearly any solvent orincorporated into a variety of inorganic and organic films. Quantum dots(QDs) can be coupled to streptavidin directly through a maleimide estercoupling reaction or to antibodies through a meleimide-thiol couplingreaction. This yields a material with a biomolecule covalently attachedon the surface, which produces conjugates with high specific activity.In some embodiments, the protein that is detected with the singlemolecule analyzer is labeled with one quantum dot. In some embodiments,the quantum dot is between 10 and 20 nm in diameter. In otherembodiments, the quantum dot is between 2 and 10 nm in diameter. Inother embodiments, the quantum dot is about 2 nm, 3 nm, 4 nm, 5 nm, 6nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 v, 16 nm, 17nm, 18 nm, 19 nm or 20 nm in diameter. Useful Quantum Dots comprise QD605, QD 610, QD 655, and QD 705. A preferred Quantum Dot is QD 605.

C. Binding Partner-Fluorescent Moiety Compositions

The labels of the invention generally contain a binding partner, e.g.,antibody, bound to a fluorescent moiety to provide the requisitefluorescence for detection and quantitation in the instruments describedherein. Any suitable combination of binding partner and fluorescentmoiety for detection in the single molecule detectors described hereinmay be used as a label in the invention. In some embodiments, theinvention provides a label for a marker of a biological state, where thelabel includes an antibody to the marker and a fluorescent moiety. Themarker may be any of the markers described above. The antibody may beany antibody as described above. A fluorescent moiety may be attachedsuch that the label is capable of emitting an average of at least about50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500, 600,700, 800, 900, or 1000, photons when simulated by a laser emitting lightat the excitation wavelength of the moiety, where the laser is focusedon a spot of not less than about 5 microns in diameter that contains thelabel, and wherein the total energy directed at the spot by the laser isno more than about 3 microJoules. In some embodiments, the fluorescentmoiety may be a fluorescent moiety that is capable of emitting anaverage of at least about 50, 100, 150, or 200 photons when simulated bya laser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the moiety, and wherein the total energy directedat the spot by the laser is no more than about 3 microJoules. Thefluorescent moiety may be a fluorescent moiety that includes one or moredye molecules with a structure that includes a substituted indolium ringsystem in which the substituent on the 3-carbon of the indolium ringcontains a chemically reactive group or a conjugated substance group.The label composition may include a fluorescent moiety that includes oneor more dye molecules selected from the group consisting of Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 700, or Alexa Fluor750. The label composition may include a fluorescent moiety thatincludes one or more dye molecules selected from the group consisting ofAlexa Fluor 488, Alexa Fluor 532, Alexa Fluor 700, or Alexa Fluor 750.The label composition may include a fluorescent moiety that includes oneor more dye molecules that are Alexa Fluor 488. The label compositionmay include a fluorescent moiety that includes one or more dye moleculesthat are Alexa Fluor 555. The label composition may include afluorescent moiety that includes one or more dye molecules that areAlexa Fluor 610. The label composition may include a fluorescent moietythat includes one or more dye molecules that are Alexa Fluor 647. Thelabel composition may include a fluorescent moiety that includes one ormore dye molecules that are Alexa Fluor 680. The label composition mayinclude a fluorescent moiety that includes one or more dye moleculesthat are Alexa Fluor 700. The label composition may include afluorescent moiety that includes one or more dye molecules that areAlexa Fluor 750.

In some embodiments the invention provides a composition for thedetection of a marker of a biological state that includes an Alexa Fluormolecule, e.g., an Alexa Fluor molecule selected from the describedgroups, such as an Alexa Fluor 647 molecule attached to an antibodyspecific for the marker. In some embodiments the composition includes anaverage of about 1 to 11, or about 2 to 10, or about 2 to 8, or about 2to 6, or about 2 to 5, or about 2 to 4, or about 3 to 10, or about 3 to8, or about 3 to 6, or about 3 to 5, or about 4 to 10, or about 4 to 8,or about 4 to 6, or about 2, 3, 4, 5, 6, or more than about 6 AlexaFluor 647 molecules attached to an antibody for the marker. In someembodiments the invention provides a composition for the detection amarker of a biological state that includes an average of about 1 to 11,or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5, orabout 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, orabout 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, orabout 2, 3, 4, 5, 6, or more than about 6 Alexa Fluor 647 moleculesattached to an antibody specific to the marker. In some embodiments theinvention provides a composition for the detection of a marker of abiological state that includes an average of about 2 to 10 Alexa Fluor647 molecules attached to an antibody specific to the marker. In someembodiments the invention provides a composition for the detection of amarker of a biological state that includes an average of about 2 to 8Alexa Fluor 647 molecules attached to an antibody specific to themarker. In some embodiments the invention provides a composition for thedetection of a marker of a biological state that includes an average ofabout 2 to 6 Alexa Fluor 647 molecules attached to an antibody specificto the marker. In some embodiments the invention provides a compositionfor the detection of a marker of a biological state that includes anaverage of about 2 to 4 Alexa Fluor 647 molecules attached to anantibody specific to the marker. In some embodiments the inventionprovides a composition for the detection of a marker of a biologicalstate that includes an average of about 3 to 8 Alexa Fluor 647 moleculesattached to an antibody specific to the marker. In some embodiments theinvention provides a composition for the detection of a marker of abiological state that includes an average of about 3 to 6 Alexa Fluor647 molecules attached to an antibody specific to the marker. In someembodiments the invention provides a composition for the detection of amarker of a biological state that includes an average of about 4 to 8Alexa Fluor 647 molecules attached to an antibody specific to themarker.

Attachment of the fluorescent moiety, or fluorescent entities that makeup the fluorescent moiety, to the binding partner, e.g., antibody, maybe by any suitable means; such methods are well-known in the art andexemplary methods are given in the Examples. In some embodiments, afterattachment of the fluorescent moiety to the binding partner to form alabel for use in the methods of the invention, and prior to the use ofthe label for labeling the protein of interest, it is useful to performa filtration step. E.g., an antibody-dye label may be filtered prior touse, e.g., through a 0.2 micron filter, or any suitable filter forremoving aggregates. Other reagents for use in the assays of theinvention may also be filtered, e.g., through a 0.2 micron filter, orany suitable filter. Without being bound by theory, it is thought thatsuch filtration removes a portion of the aggregates of the, e.g.,antibody-dye labels. As such aggregates can bind as a unit to theprotein of interest, but upon release in elution buffer are likely todisaggregate, false positives may result; i.e., several labels will bedetected from an aggregate that has bound to only a single proteinmolecule of interest. Regardless of theory, filtration has been found toreduce false positives in the subsequent assay and to improve accuracyand precision.

It will be appreciated that immunoassays often employ a sandwich format,in which binding partner pairs, e.g., antibodies, to the same molecule,e.g., a marker, are used. The invention also encompasses binding partnerpairs, e.g., antibodies, wherein both antibodies are specific to thesame molecule, e.g., the same marker, and wherein at least one member ofthe pair is a label as described herein. Thus, for any label thatincludes a binding-partner and a fluorescent moiety, the invention alsoencompasses a pair of binding partners wherein the first bindingpartner, e.g., antibody, is part of the label, and the second bindingpartner, e.g., antibody, is, typically, unlabeled and serves as acapture binding partner. In addition, binding partner pairs arefrequently used in FRET assays. FRET assays useful in the invention aredisclosed in U.S. patent application Ser. No. 11/048,660, incorporatedby reference herein in its entirety, and the present invention alsoencompasses binding partner pairs, each of which includes a FRET label.

IV. HIGHLY SENSITIVE ANALYSIS OF MOLECULES

In one aspect, the invention provides a method for determining thepresence or absence of a single molecule, e.g., a molecule of a markerof a biological state, in a sample, by i) labeling the molecule ifpresent, with a label; and ii) detecting the presence or absence of thelabel, where the detection of the presence of the label indicates thepresence of the single molecule in the sample. In some embodiments, themethod is capable of detecting the molecule at a limit of detection ofless than about 100, 80, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5,4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01,0.005, or 0.001 femtomolar. In some embodiments, the method is capableof detecting the molecule at a limit of detection of less than about 100femtomolar. In some embodiments, the method is capable of detecting themolecule at a limit of detection of less than about 10 femtomolar. Insome embodiments, the method is capable of detecting the molecule at alimit of detection of less than about 1 femtomolar. In some embodiments,the method is capable of detecting the molecule at a limit of detectionof less than about 0.1 femtomolar. In some embodiments, the method iscapable of detecting the molecule at a limit of detection of less thanabout 0.01 femtomolar. In some embodiments, the method is capable ofdetecting the molecule at a limit of detection of less than about 0.001femtomolar. Detection limits may be determined by use of an appropriatestandard, e.g., National Institute of Standards and Technology referencestandard material.

The methods also provide methods of determining a concentration of amolecule, e.g., a marker indicative of a biological state, in a sampleby detecting single molecules of the molecule in the sample. The“detecting” of a single molecule includes detecting the moleculedirectly or indirectly. In the case of indirect detection, labels thatcorrespond to single molecules, e.g., labels attached to the singlemolecules, can be detected.

In some embodiments, the invention provides a method for determining thepresence or absence of a single molecule of a protein in a biologicalsample, comprising labeling said molecule with a label and detecting thepresence or absence of said label in a single molecule detector, whereinsaid label comprises a fluorescent moiety that is capable of emitting atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, wherein the laser is focused on aspot not less than about 5 microns in diameter that contains the moiety,and wherein the total energy directed at the spot by the laser is nomore than about 3 microJoules. The single molecule detector may, in someembodiments, comprise not more than one interrogation space. The limitof detection of the single molecule in the sample may be less than about10, 1, 0.1, 0.01, or 0.001 femtomolar. In some embodiments, the limit ofdetection is less than about 1 femtomolar. The detecting may comprisedetecting electromagnetic radiation emitted by said fluorescent moiety.The method may further comprise exposing said fluorescent moiety toelectromagnetic radiation, e.g., electromagnetic radiation provided by alaser, such as a laser with a power output of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mW. In some embodiments,the laser stimulus provides light to the interrogation space for betweenabout 10-1000 microseconds, or about 1000, 250, 100, 50, 25 or 10microseconds. In some embodiments, the label further comprises a bindingpartner specific for binding said molecule, such as an antibody. In someembodiments, the fluorescent moiety comprises a fluorescent dyemolecule, such as a dye molecule that comprises at least one substitutedindolium ring system in which the substituent on the 3-carbon of theindolium ring contains a chemically reactive group or a conjugatedsubstance. In some embodiments, the dye molecule is an AlexFluormolecule selected from the group consisting of Alexa Fluor 488, AlexaFluor 532, Alexa Fluor 647, Alexa Fluor 680 or Alexa Fluor 700. In someembodiments, the dye molecule is an Alexa Fluor 647 dye molecule. Insome embodiments, the fluorescent moiety comprises a plurality of AlexaFluor 647 molecules. In some embodiments, the plurality of Alexa Fluor647 molecules comprises about 2-4 Alexa Fluor 647 molecules, or about3-6 Alexa Fluor 647 molecules. In some embodiments, the fluorescentmoiety is a quantum dot. The method may further comprise measuring theconcentration of said protein in the sample.

In some embodiments, detecting the presence or absence of said labelcomprises: (i) passing a portion of said sample through an interrogationspace; (ii) subjecting said interrogation space to exposure toelectromagnetic radiation, said electromagnetic radiation beingsufficient to stimulate said fluorescent moiety to emit photons, if saidlabel is present; and (iii) detecting photons emitted during saidexposure of step (ii). The method may further comprise determining abackground photon level in said interrogation space, wherein saidbackground level represents the average photon emission of theinterrogation space when it is subjected to electromagnetic radiation inthe same manner as in step (ii), but without label in the interrogationspace. The method may further comprise comparing the amount of photonsdetected in step (iii) to a threshold photon level, wherein saidthreshold photon level is a function of said background photon level,wherein an amount of photons detected in step (iii) greater that thethreshold level indicates the presence of said label, and an amount ofphotons detected in step (iii) equal to or less than the threshold levelindicates the absence of said label.

A. Sample

The sample may be any suitable sample. Typically, the sample is abiological sample, e.g., a biological fluid. Such fluids include,without limitation, bronchoalveolar lavage fluid (BAL), blood, serum,plasma, urine, nasal swab, cerebrospinal fluid, pleural fluid, synovialfluid, peritoneal fluid, amniotic fluid, gastric fluid, lymph fluid,interstitial fluid, tissue homogenate, cell extracts, saliva, sputum,stool, physiological secretions, tears, mucus, sweat, milk, semen,seminal fluid, vaginal secretions, fluid from ulcers and other surfaceeruptions, blisters, and abscesses, and extracts of tissues includingbiopsies of normal, malignant, and suspect tissues or any otherconstituents of the body which may contain the target particle ofinterest. Other similar specimens such as cell or tissue culture orculture broth are also of interest.

In some embodiments, the sample is a blood sample. In some embodimentsthe sample is a plasma sample. In some embodiments the sample is a serumsample. In some embodiments, the sample is a urine sample. In someembodiments, the sample is a nasal swab. In some embodiments, the sampleis a cell lysate. In some embodiments, the sample is a tissue sample.

B. Sample preparation

In general, any method of sample preparation may be used that produces alabel corresponding to a molecule of interest, e.g., a marker of abiological state to be measured, where the label is detectable in theinstruments described herein. As is known in the art, sample preparationin which a label is added to one or more molecules may be performed in ahomogeneous or heterogeneous format. In some embodiments, the samplepreparation is formed in a homogenous format. In analyzer systemsemploying a homogenous format, unbound label is not removed from thesample. See, e.g., U.S. patent application Ser. No. 11/048,660. In someembodiments, the particle or particles of interest are labeled byaddition of labeled antibody or antibodies that bind to the particle orparticles of interest.

In some embodiments, a heterogeneous assay format is used, where,typically, a step is employed for removing unbound label. Such assayformats are well-known in the art. One particularly useful assay formatis a sandwich assay, e.g., a sandwich immunoassay. In this format, themolecule of interest, e.g., marker of a biological state, is captured,e.g., on a solid support, using a capture binding partner. Unwantedmolecules and other substances may then optionally be washed away,followed by binding of a label comprising a detection binding partnerand a detectable label, e.g., fluorescent moiety. Further washes removeunbound label, then the detectable label is released, usually though notnecessarily still attached to the detection binding partner. Inalternative embodiments, sample and label are added to the capturebinding partner without a wash in between, e.g., at the same time. Othervariations will be apparent to one of skill in the art.

In some embodiments, the method for detecting the molecule of interest,e.g., marker of a biological state, uses a sandwich assay withantibodies, e.g., monoclonal antibodies as capture binding partners. Themethod comprises binding molecules in a sample to a capture antibodythat is immobilized on a binding surface, and binding the labelcomprising a detection antibody to the molecule to form a “sandwich”complex. The label comprises the detection antibody and a fluorescentmoiety, as described herein, which is detected, e.g., using the singlemolecule analyzers of the invention. Both the capture and detectionantibodies specifically bind the molecule. Many examples of sandwichimmunoassays are known, and some are described in U.S. Pat. No.4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al.,both of which are incorporated herein by reference. Further examplesspecific to specific markers are described in the Examples.

The capture binding partner may be attached to a solid support, e.g., amicrotiter plate or paramagnetic beads. In some embodiments, theinvention provides a binding partner for a molecule of interest, e.g.,marker of a biological state, attached to a paramagnetic bead. Anysuitable binding partner that is specific for the molecule that it iswished to capture may be used. The binding partner may be an antibody,e.g., a monoclonal antibody. Production and sources of antibodies aredescribed elsewhere herein. It will be appreciated that antibodiesidentified herein as useful as a capture antibody may also be useful asdetection antibodies, and vice versa.

The attachment of the binding partner, e.g., antibody, to the solidsupport may be covalent or noncovalent. In some embodiments, theattachment is noncovalent. An example of a noncovalent attachmentwell-known in the art is biotin-avidin/streptavidin interactions. Thus,in some embodiments, a solid support, e.g., a microtiter plate or aparamagnetic bead, is attached to the capture binding partner, e.g.,antibody, through noncovalent attachment, e.g.,biotin-avidin/streptavidin interactions. In some embodiments, theattachment is covalent. Thus, in some embodiments, a solid support,e.g., a microtiter plate or a paramagnetic bead, is attached to thecapture binding partner, e.g., antibody, through covalent attachment.

The capture antibody can be covalently attached in an orientation thatoptimizes the capture of the molecule of interest. For example, in someembodiments, a binding partner, e.g., an antibody, is attached in aorientated manner to a solid support, e.g., a microtiter plate or aparamagnetic microparticle.

An exemplary protocol for oriented attachment of an antibody to a solidsupport is as follows: IgG is dissolved in 0.1M sodium acetate buffer,pH 5.5 to a final concentration of 1 mg/ml. An equal volume of ice-cold20 mM sodium periodate in 0.1M sodium acetate, pH 5.5 is added. The IgGis allowed to oxidize for ½ hour on ice. Excess periodate reagent isquenched by the addition of 0.15 volume of 1M glycerol. Low molecularweight byproducts of the oxidation reaction are removed byultrafiltration. The oxidized IgG fraction is diluted to a suitableconcentration (typically 0.5 micrograms IgG per ml) and reacted withhydrazide-activated multiwell plates for at least two hours at roomtemperature. Unbound IgG is removed by washing the multiwell plate withborate buffered saline or another suitable buffer. The plate may bedried for storage, if desired. A similar protocol may be followed formicrobeads if the material of the microbead is suitable for suchattachment.

In some embodiments, the solid support is a microtiter plate. In someembodiments, the solid support is a paramagnetic bead. An exemplaryparamagnetic bead is Streptavidin C1 (Dynal, 650.01-03). Other suitablebeads will be apparent to those of skill in the art. Methods forattachment of antibodies to paramagnetic beads are well-known in theart. One example is given in Example 2.

The molecule of interest is contacted with the capture binding partner,e.g., capture antibody immobilized on a solid support. Some samplepreparation may be used; e.g., preparation of serum from blood samplesor concentration procedures before the sample is contacted with thecapture antibody. Protocols for binding of proteins in immunoassays arewell-known in the art and are included in the Examples.

The time allowed for binding will vary depending on the conditions; itwill be apparent that shorter binding times are desirable in somesettings, especially in a clinical setting. The use of, e.g.,paramagnetic beads can reduce the time required for binding. In someembodiments, the time allowed for binding of the molecule of interest tothe capture binding partner, e.g., an antibody, is less that about 12,10, 8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25,20, 15, 10, or 5 minutes. In some embodiments, the time allowed forbinding of the molecule of interest to the capture binding partner,e.g., an antibody, is less than about 60 minutes. In some embodiments,the time allowed for binding of the molecule of interest to the capturebinding partner, e.g., an antibody, is less than about 40 minutes. Insome embodiments, the time allowed for binding of the molecule ofinterest to the capture binding partner, e.g., an antibody, is less thanabout 30 minutes. In some embodiments, the time allowed for binding ofthe molecule of interest to the capture binding partner, e.g., anantibody, is less than about 20 minutes. In some embodiments, the timeallowed for binding of the molecule of interest to the capture bindingpartner, e.g., an antibody, is less than about 15 minutes. In someembodiments, the time allowed for binding of the molecule of interest tothe capture binding partner, e.g., an antibody, is less than about 10minutes. In some embodiments, the time allowed for binding of themolecule of interest to the capture binding partner, e.g., an antibody,is less than about 5 minutes.

In some embodiments, following the binding of particles of the moleculeof interest to the capture binding partner, e.g., a capture antibody,particles that bound nonspecifically, as well as other unwantedsubstances in the sample, are washed away leaving substantially onlyspecifically bound particles of the molecule of interest. In otherembodiments, no wash is used between additions of sample and label,which can reduce sample preparation time. Thus, in some embodiments, thetime allowed for both binding of the molecule of interest to the capturebinding partner, e.g., an antibody, and binding of the label to themolecule of interest, is less that about 12, 10, 8, 6, 4, 3, 2, or 1hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or minutes. Insome embodiments, the time allowed for both binding of the molecule ofinterest to the capture binding partner, e.g., an antibody, and bindingof the label to the molecule of interest, is less that about 60 minutes.In some embodiments, the time allowed for both binding of the moleculeof interest to the capture binding partner, e.g., an antibody, andbinding of the label to the molecule of interest, is less that about 50minutes. In some embodiments, the time allowed for both binding of themolecule of interest to the capture binding partner, e.g., an antibody,and binding of the label to the molecule of interest, is less than about40 minutes. In some embodiments, the time allowed for both binding ofthe molecule of interest to the capture binding partner, e.g., anantibody, and binding of the label to the molecule of interest, is lessthan about 30 minutes. In some embodiments, the time allowed for bothbinding of the molecule of interest to the capture binding partner,e.g., an antibody, and binding of the label to the molecule of interest,is less than about 20 minutes. In some embodiments, the time allowed forboth binding of the molecule of interest to the capture binding partner,e.g., an antibody, and binding of the label to the molecule of interest,is less than about 15 minutes. In some embodiments, the time allowed forboth binding of the molecule of interest to the capture binding partner,e.g., an antibody, and binding of the label to the molecule of interest,is less than about 10 minutes. In some embodiments, the time allowed forboth binding of the molecule of interest to the capture binding partner,e.g., an antibody, and binding of the label to the molecule of interest,is less than about 5 minutes.

Some immunoassay diagnostic reagents, including the capture and signalantibodies used to measure the molecule of interest, can be derived fromanimal sera. Endogenous human heterophilic antibodies, or humananti-animal antibodies, which have the ability to bind toimmunoglobulins of other species, are present in the serum or plasma ofmore than 10% of patients. These circulating heterophilic antibodies caninterfere with immunoassay measurements. In sandwich immunoassays, theseheterophilic antibodies can either bridge the capture and detection(diagnostic) antibodies, thereby producing a false-positive signal, orthey can block the binding of the diagnostic antibodies, therebyproducing a false-negative signal. In competitive immunoassays, theheterophilic antibodies can bind to the analytic antibody and inhibitits binding to the molecule of interest. They can also either block oraugment the separation of the antibody-molecule of interest complex fromfree molecule of interest, especially when antispecies antibodies areused in the separation systems. Therefore, the impact of theseheterophilic antibody interferences is difficult to predict and it canbe advantageous to block the binding of heterophilic antibodies. In someembodiments of the invention, the immunoassay includes the step ofdepleting the sample of heterophilic antibodies using one or moreheterophilic antibody blockers. Methods for removing heterophilicantibodies from samples to be tested in immunoassays are known andinclude: heating the specimen in a sodium acetate buffer, pH 5.0, for 15minutes at 90° C. and centrifuging at 1200 g for 10 minutes;precipitating the heterophilic immunoglobulins using polyethylene glycol(PEG); immunoextracting the interfering heterophilic immunoglobulinsfrom the specimen using protein A or protein G; or adding nonimmunemouse IgG. Embodiments of the methods of the invention contemplatepreparing the sample prior to analysis with the single moleculedetector. The appropriateness of the method of pretreatment can bedetermined. Biochemicals to minimize immunoassay interference caused byheterophilic antibodies are commercially available. For example, aproduct called MAK33, which is an IgG1 monoclonal antibody to h-CK-MM,can be obtained from Boehringer Mannheim. The MAK33 plus productcontains a combination of IgG1 and IgG1-Fab. polyMAK33 contains IgG1-Fabpolymerized with IgG1, and the polyMAC 2b/2a contains IgG2a-Fabpolymerized with IgG2b. Bioreclamation Inc., East Meadow, N.Y., marketsa second commercial source of biochemicals to neutralize heterophilicantibodies known as Immunoglobulin Inhibiting Reagent. This product is apreparation of immunoglobulins (IgG and IgM) from multiple species,mainly murine IgG2a, IgG2b, and IgG3 from Balb/c mice. In someembodiments the heterophilic antibody can be immunoextracted from thesample using methods known in the art, e.g., depleting the sample of theheterophilic antibody by binding the interfering antibody to protein Aor protein G. In some embodiments, the heterophilic antibody can beneutralized using one or more heterophilic antibody blockers.Heterophilic blockers can be selected from the group consisting ofanti-isotype heterophilic antibody blockers, anti-idiotype heterophilicantibody blockers, and anti-anti-idiotype heterophilic antibodyblockers. In some embodiments, a combination of heterophilic antibodyblockers can be used.

Label is added either with or following the addition of sample andwashing. Protocols for binding antibodies and other immunolabels toproteins and other molecules are well-known in the art. If the labelbinding step is separate from that of capture binding, the time allowedfor label binding can be important, e.g., in clinical applications orother time sensitive settings. In some embodiments, the time allowed forbinding of the molecule of interest to the label, e.g., an antibody-dye,is less than about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than about60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 60 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 50 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 40 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 30 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 20 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 15 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 10 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the label, e.g.,an antibody-dye, is less than about 5 minutes. Excess label is removedby washing.

In some embodiments, the label is not eluted from the protein ofinterest. In other embodiments, the label is eluted from the protein ofinterest. Preferred elution buffers are effective in releasing the labelwithout generating significant background. It is useful if the elutionbuffer is bacteriostatic. Elution buffers used in the invention cancomprise a chaotrope, a buffer, an albumin to coat the surface of themicrotiter plate, and a surfactant selected so as to produce arelatively low background. The chaotrope can comprise urea, aguanidinium compound, or other useful chaotropes. The buffer cancomprise borate buffered saline, or other useful buffers. The proteincarrier can comprise, e.g., an albumin, such as human, bovine, or fishalbumin, an IgG, or other useful carriers. The surfactant can comprisean ionic or nonionic detergent including Tween 20, Triton X-100, sodiumdodecyl sulfate (SDS), and others.

In another embodiment, the solid phase binding assay can be acompetitive binding assay. One such method is as follows. First, acapture antibody immobilized on a binding surface is competitively boundby i) a molecule of interest, e.g., marker of a biological state, in asample, and ii) a labeled analog of the molecule comprising a detectablelabel (the detection reagent). Second, the amount of the label using asingle molecule analyzer is measured. Another such method is as follows.First, an antibody having a detectable label (the detection reagent) iscompetitively bound to i) a molecule of interest, e.g., marker of abiological state in a sample, and ii) an analog of the molecule that isimmobilized on a binding surface (the capture reagent). Second, theamount of the label using a single molecule analyzer is measured. An“analog of a molecule” refers, herein, to a species that competes with amolecule for binding to a capture antibody. Examples of competitiveimmunoassays are disclosed in U.S. Pat. No. 4,235,601 to Deutsch et al.,U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 toBuechler et al., all of which are incorporated herein by reference.

C. Detection of Molecule of Interest and Determination of Concentration

Following elution, the label is run through a single molecule detectorin, e.g., the elution buffer. A processing sample may contain no label,a single label, or a plurality of labels. The number of labelscorresponds or is proportional to (if dilutions or fractions of samplesare used) the number of molecules of the molecule of interest, e.g.,marker of a biological state captured during the capture step.

Any suitable single molecule detector capable of detecting the labelused with the molecule of interest may be used. Suitable single moleculedetectors are described herein. Typically the detector will be part of asystem that includes an automatic sampler for sampling prepared samples,and, optionally, a recovery system to recover samples.

In some embodiments, the processing sample is analyzed in a singlemolecule analyzer that utilizes a capillary flow system, and thatincludes a capillary flow cell, a laser to illuminate an interrogationspace in the capillary through which processing sample is passed, adetector to detect radiation emitted from the interrogation space, and asource of motive force to move a processing sample through theinterrogation space. In some embodiments, the single molecule analyzerfurther comprises a microscope objective lens that collects lightemitted from processing sample as it passes through the interrogationspace, e.g., a high numerical aperture microscope objective. In someembodiments, the laser and detector are in a confocal arrangement. Insome embodiments, the laser is a continuous wave laser. In someembodiments, the detector is an avalanche photodiode detector. In someembodiments, the source of motive force is a pump to provide pressure.In some embodiments, the invention provides an analyzer system thatincludes a sampling system capable of automatically sampling a pluralityof samples providing a fluid communication between a sample containerand the interrogation space. In some embodiments, the interrogationspace has a volume of between about 0.001 and 500 pL, or between about0.01 pL and 100 pL, or between about 0.01 pL and 10 pL, or between about0.01 pL and 5 pL, or between about 0.01 pL and 0.5 pL, or between about0.02 pL and about 300 pL, or between about 0.02 pL and about 50 pL orbetween about 0.02 pL and about 5 pL or between about 0.02 pL and about0.5 pL or between about 0.02 pL and about 2 pL, or between about 0.05 pLand about 50 pL, or between about 0.05 pL and about 5 pL, or betweenabout 0.05 pL and about 0.5 pL, or between about 0.05 pL and about 0.2pL, or between about 0.1 pL and about 25 pL. In some embodiments, theinterrogation space has a volume between about 0.004 pL and 100 pL. Insome embodiments, the interrogation space has a volume between about0.02 pL and 50 pL. In some embodiments, the interrogation space has avolume between about 0.001 pL and 10 pL. In some embodiments, theinterrogation space has a volume between about 0.001 pL and 10 pL. Insome embodiments, the interrogation space has a volume between about0.01 pL and 5 pL. In some embodiments, the interrogation space has avolume between about 0.02 pL and about 5 pL. In some embodiments, theinterrogation space has a volume between about 0.05 pL and 5 pL. In someembodiments, the interrogation space has a volume between about 0.05 pLand 10 pL. In some embodiments, the interrogation space has a volumebetween about 0.5 pL and about 5 pL. In some embodiments, theinterrogation space has a volume between about 0.02 pL and about 0.5 pL.

In some embodiments, the interrogation space has a volume of more thanabout 1 μm³, more than about 2 μm³, more than about 3 μm³, more thanabout 4 μm³, more than about 5 μm³, more than about 10 μm³, more thanabout 15 μm³, more than about 30 μm³, more than about 50 μm³, more thanabout 75 μm³, more than about 100 μm³, more than about 150 μm³, morethan about 200 μm³, more than about 250 μm³, more than about 300 μm³,more than about 400 μm³, more than about 500 μm³, more than about 550μm³, more than about 600 m³, more than about 750 m³, more than about1000 m³, more than about 2000 m³, more than about 4000 μm³, more thanabout 6000 μm³, more than about 8000 μm³, more than about 10000 μm³,more than about 12000 μm³, more than about 13000 μm³, more than about14000 μm³, more than about 15000 μm³, more than about 20000 μm³, morethan about 30000 μm³, more than about 40000 m³, or more than about 50000μm³. In some embodiments, the interrogation space is of a volume lessthan about 50000 μm³, less than about 40000 μm³, less than about 30000μm³, less than about 20000 μm³, less than about 15000 μm³, less thanabout 14000 μm³, less than about 13000 μm³, less than about 12000 μm³,less than about 11000 μm³, less than about 9500 μm³, less than about8000 μm³, less than about 6500 μm³, less than about 6000 μm³, less thanabout 5000 μm³, less than about 4000 μm³, less than about 3000 μm³, lessthan about 2500 μm³, less than about 2000 μm³, less than about 1500 μm³,less than about 1000 μm³, less than about 800 μm³, less than about 600μm³, less than about 400 μm³, less than about 200 μm³, less than about100 μm³, less than about 75 μm³, less than about 50 μm³, less than about25 μm³, less than about 20 μm³, less than about 15 μm³, less than about14 μm³, less than about 13 μm³, less than about 12 μm³, less than about11 μm³, less than about 10 μm³, less than about 5 μm³, less than about 4μm³, less than about 3 μm³, less than about 2 μm³, or less than about 1μm³. In some embodiments, the volume of the interrogation space isbetween about 1 μm³ and about 10000 μm³. In some embodiments, theinterrogation space is between about 1 μm³ and about 1000 μm³. In someembodiments, the interrogation space is between about 1 μm³ and about100 μm³. In some embodiments, the interrogation space is between about 1μm³ and about 50 μm³. In some embodiments, the interrogation space isbetween about 1 μm³ and about 10 μm³. In some embodiments, theinterrogation space is between about 2 μm³ and about 10 μm³. In someembodiments, the interrogation space is between about 3 μm³ and about 7μm³.

In some embodiments, the single molecule detector used in the methods ofthe invention utilizes a capillary flow system, and includes a capillaryflow cell, a continuous wave laser to illuminate an interrogation spacein the capillary through which processing sample is passed, a highnumerical aperture microscope objective lens that collects light emittedfrom processing sample as it passes through the interrogation space, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a pump to provide pressure to move a processingsample through the interrogation space, where the interrogation space isbetween about 0.02 pL and about 50 pL. In some embodiments, the singlemolecule detector used in the methods of the invention utilizes acapillary flow system, and includes a capillary flow cell, a continuouswave laser to illuminate an interrogation space in the capillary throughwhich processing sample is passed, a high numerical aperture microscopeobjective lens that collects light emitted from processing sample as itpasses through the interrogation space wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a pump toprovide pressure to move a processing sample through the interrogationspace, where the interrogation space is between about 0.004 pL and about100 pL. In some embodiments, the single molecule detector used in themethods of the invention utilizes a capillary flow system, and includesa capillary flow cell, a continuous wave laser to illuminate aninterrogation space in the capillary through which processing sample ispassed, a high numerical aperture microscope objective lens thatcollects light emitted from processing sample as it passes through theinterrogation space wherein the lens has a numerical aperture of atleast about 0.8, an avalanche photodiode detector to detect radiationemitted from the interrogation space, and a pump to provide pressure tomove a processing sample through the interrogation space, where theinterrogation space is between about 0.05 pL and about 10 pL. In someembodiments, the single molecule detector used in the methods of theinvention utilizes a capillary flow system, and includes a capillaryflow cell, a continuous wave laser to illuminate an interrogation spacein the capillary through which processing sample is passed, a highnumerical aperture microscope objective lens that collects light emittedfrom processing sample as it passes through the interrogation spacewherein the lens has a numerical aperture of at least about 0.8, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a pump to provide pressure to move a processingsample through the interrogation space, where the interrogation space isbetween about 0.05 pL and about 5 pL. In some embodiments, the singlemolecule detector used in the methods of the invention utilizes acapillary flow system, and includes a capillary flow cell, a continuouswave laser to illuminate an interrogation space in the capillary throughwhich processing sample is passed, a high numerical aperture microscopeobjective lens that collects light emitted from processing sample as itpasses through the interrogation space wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a pump toprovide pressure to move a processing sample through the interrogationspace, where the interrogation space is between about 0.5 pL and about 5pL. In any of these embodiments the analyzer may contain not more thanone interrogation space.

In some embodiments, the single molecule detector comprises a scanninganalyzer system, as disclosed in U.S. patent application Ser. No.12/338,955, filed Dec. 18, 2008 and entitled “Scanning Analyzer forSingle Molecule Detection and Methods of Use.” In some embodiments, thesingle molecule detector used in the methods of the invention uses asample plate, a continuous wave laser directed toward a sample plate inwhich the sample is contained, a high numerical aperture microscopeobjective lens that collects light emitted from the sample asinterrogation space is translated through the sample, wherein the lenshas a numerical aperture of at least about 0.8, an avalanche photodiodedetector to detect radiation emitted from the interrogation space, and ascan motor with a moveable mirror to translate the interrogation spacethrough the sample wherein the interrogation space is between about 1μm³ and about 10000 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 1 μm³ and about 1000 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample,wherein the lens has a numerical aperture of at least about 0.8, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 1 μm³ and about 100 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 1 μm³ and about 10 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample,wherein the lens has a numerical aperture of at least about 0.8, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 2 μm³ and about 10 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 2 μm³ and about 8 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample,wherein the lens has a numerical aperture of at least about 0.8, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 3 μm³ and about 7 μm³. In any of these embodiments, the analyzercan contain only one interrogation space.

In other embodiments, the single molecule detector used in the methodsof the invention uses a sample plate, a continuous wave laser directedtoward a sample plate in which the sample is contained, a high numericalaperture microscope objective lens that collects light emitted from thesample as interrogation space is translated through the sample, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor with a moveable mirror totranslate the interrogation space through the sample wherein theinterrogation space is between about 1 μm³ and about 10000 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 1 μm³ and about 1000 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 1 μm³ and about 100 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 1 μm³ and about 10 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 2 μm³ and about 10 μm³. In someembodiments, the single molecule detector used in the methods of theinvention uses a sample plate, a continuous wave laser directed towardan interrogation space located within the sample, a high numericalaperture microscope objective lens that collects light emitted from thesample as the interrogation space is translated through the sample, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a scan motor for translating the interrogationspace through the sample, wherein the interrogation space is betweenabout 2 μm³ and about 8 μm³. In some embodiments, the single moleculedetector used in the methods of the invention uses a sample plate, acontinuous wave laser directed toward an interrogation space locatedwithin the sample, a high numerical aperture microscope objective lensthat collects light emitted from the sample as the interrogation spaceis translated through the sample, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a scan motorfor translating the interrogation space through the sample, wherein theinterrogation space is between about 3 μm³ and about 7 μm³. In any ofthese embodiments, the analyzer can contain only one interrogationspace.

In some embodiments, the single molecule detector is capable ofdetermining a concentration for a molecule of interest in a sample wheresample may range in concentration over a range of at least about100-fold, or 1000-fold, or 10,000-fold, or 100,000-fold, or300,000-fold, or 1,000,000-fold, or 10,000,000-fold, or 30,000,000-fold.

In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 50%,40%, 30%, 20%, 15%, or 10% in concentration of an analyte between afirst sample and a second sample that are introduced into the detector,where the volume of the first sample and said second sample introducedinto the analyzer is less than about 100, 90, 80, 70, 60, 50, 40, 30,20, 15, 10, 5, 4, 3, 2, or 1 μl, and wherein the analyte is present at aconcentration of less than about 100, 90, 80, 70, 60, 50, 40, 30, 20,15, 10, 5, 4, 3, 2, or 1 femtomolar. In some embodiments, the methods ofthe invention utilize a single molecule detector capable detecting adifference of less than about 50% in concentration of an analyte betweena first sample and a second sample that are introduced into thedetector, where the volume of the first sample and said second sampleintroduced into the analyzer is less than about 100 μl, and wherein theanalyte is present at a concentration of less than about 100 femtomolar.In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 40%in concentration of an analyte between a first sample and a secondsample that are introduced into the detector, where the volume of thefirst sample and said second sample introduced into the analyzer is lessthan about 50 μl, and wherein the analyte is present at a concentrationof less than about 50 femtomolar. In some embodiments, the methods ofthe invention utilize a single molecule detector capable detecting adifference of less than about 20% in concentration of an analyte betweena first sample and a second sample that are introduced into thedetector, where the volume of the first sample and said second sampleintroduced into the analyzer is less than about 20 μl, and wherein theanalyte is present at a concentration of less than about 20 femtomolar.In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 20%in concentration of an analyte between a first sample and a secondsample that are introduced into the detector, where the volume of thefirst sample and said second sample introduced into the analyzer is lessthan about 10 μl, and wherein the analyte is present at a concentrationof less than about 10 femtomolar. In some embodiments, the methods ofthe invention utilize a single molecule detector capable detecting adifference of less than about 20% in concentration of an analyte betweena first sample and a second sample that are introduced into thedetector, where the volume of the first sample and said second sampleintroduced into the analyzer is less than about 5 μl, and wherein theanalyte is present at a concentration of less than about 5 femtomolar.In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 20%in concentration of an analyte between a first sample and a secondsample that are introduced into the detector, where the volume of thefirst sample and said second sample introduced into the analyzer is lessthan about 5 μl, and wherein the analyte is present at a concentrationof less than about 50 femtomolar.

The single molecule detector and systems are described in more detailbelow. Further embodiments of single molecule analyzers useful in themethods of the invention, such as detectors with more than oneinterrogation window, detectors utilize electrokinetic orelectrophoretic flow, and the like, may be found in U.S. patentapplication Ser. No. 11/048,660, incorporated by reference herein in itsentirety.

Between runs the instrument may be washed. A wash buffer that maintainsthe salt and surfactant concentrations of the sample may be used in someembodiments to maintain the conditioning of the capillary; i.e., to keepthe capillary surface relatively constant between samples to reducevariability.

A feature that contributes to the extremely high sensitivity of theinstruments and methods of the invention is the method of detecting andcounting labels, which, in some embodiments, are attached to singlemolecules to be detected or, more typically, correspond to a singlemolecule to be detected. Briefly, the processing sample flowing throughthe capillary or contained on a sample plate is effectively divided intoa series of detection events, by subjecting a given interrogation spaceof the capillary to EM radiation from a laser that emits light at anappropriate excitation wavelength for the fluorescent moiety used in thelabel for a predetermined period of time, and detecting photons emittedduring that time. Each predetermined period of time is a “bin.” If thetotal number of photons detected in a given bin exceeds a predeterminedthreshold level, a detection event is registered for that bin, i.e., alabel has been detected. If the total number of photons is not at thepredetermined threshold level, no detection event is registered. In someembodiments, processing sample concentration is dilute enough that, fora large percentage of detection events, the detection event representsonly one label passing through the window, which corresponds to a singlemolecule of interest in the original sample, that is, few detectionevents represent more than one label in a single bin. In someembodiments, further refinements are applied to allow greaterconcentrations of label in the processing sample to be detectedaccurately, i.e., concentrations at which the probability of two or morelabels being detected as a single detection event is no longerinsignificant.

Although other bin times can be used without departing from the scope ofthe present invention, in some embodiments the bin times are selected inthe range of about 1 microsecond to about 5 ms. In some embodiments, thebin time is more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750,800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. In someembodiments, the bin time is less than about 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600,700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. Insome embodiments, the bin time is about 1 to 1000 microseconds. In someembodiments, the bin time is about 1 to 750 microseconds. In someembodiments, the bin time is about 1 to 500 microseconds. In someembodiments, the bin time is about 1 to 250 microseconds. In someembodiments, the bin time is about 1 to 100 microseconds. In someembodiments, the bin time is about 1 to 50 microseconds. In someembodiments, the bin time is about 1 to 40 microseconds. In someembodiments, the bin time is about 1 to 30 microseconds. In someembodiments, the bin time is about 1 to 25 microseconds. In someembodiments, the bin time is about 1 to 20 microseconds. In someembodiments, the bin time is about 1 to 10 microseconds. In someembodiments, the bin time is about 1 to 7.5 microseconds. In someembodiments, the bin time is about 1 to 5 microseconds. In someembodiments, the bin time is about 5 to 500 microseconds. In someembodiments, the bin time is about 5 to 250 microseconds. In someembodiments, the bin time is about 5 to 100 microseconds. In someembodiments, the bin time is about 5 to 50 microseconds. In someembodiments, the bin time is about 5 to 20 microseconds. In someembodiments, the bin time is about 5 to 10 microseconds. In someembodiments, the bin time is about 10 to 500 microseconds. In someembodiments, the bin time is about 10 to 250 microseconds. In someembodiments, the bin time is about 10 to 100 microseconds. In someembodiments, the bin time is about 10 to 50 microseconds. In someembodiments, the bin time is about 10 to 30 microseconds. In someembodiments, the bin time is about 10 to 20 microseconds. In someembodiments, the bin time is about 1 microsecond. In some embodiments,the bin time is about 2 microseconds. In some embodiments, the bin timeis about 3 microseconds. In some embodiments, the bin time is about 4microseconds. In some embodiments, the bin time is about 5 microseconds.In some embodiments, the bin time is about 6 microseconds. In someembodiments, the bin time is about 7 microseconds. In some embodiments,the bin time is about 8 microseconds. In some embodiments, the bin timeis about 9 microseconds. In some embodiments, the bin time is about 10microseconds. In some embodiments, the bin time is about 11microseconds. In some embodiments, the bin time is about 12microseconds. In some embodiments, the bin time is about 13microseconds. In some embodiments, the bin time is about 14microseconds. In some embodiments, the bin time is about 5 microseconds.In some embodiments, the bin time is about 15 microseconds. In someembodiments, the bin time is about 16 microseconds. In some embodiments,the bin time is about 17 microseconds. In some embodiments, the bin timeis about 18 microseconds. In some embodiments, the bin time is about 19microseconds. In some embodiments, the bin time is about 20microseconds. In some embodiments, the bin time is about 25microseconds. In some embodiments, the bin time is about 30microseconds. In some embodiments, the bin time is about 40microseconds. In some embodiments, the bin time is about 50microseconds. In some embodiments, the bin time is about 100microseconds. In some embodiments, the bin time is about 250microseconds. In some embodiments, the bin time is about 500microseconds. In some embodiments, the bin time is about 750microseconds. In some embodiments, the bin time is about 1000microseconds.

In some embodiments, determining the concentration of a particle-labelcomplex in a sample comprises determining the background noise level. Insome embodiments, the background noise level is determined from the meannoise level, or the root-mean-square noise. In other cases, a typicalnoise value or a statistical value is chosen. In most cases, the noiseis expected to follow a Poisson distribution.

Thus, as a label is encountered in the interrogation space, it isirradiated by the laser beam to generate a burst of photons. The photonsemitted by the label are discriminated from background light orbackground noise emission by considering only the bursts of photons thathave energy above a predetermined threshold energy level which accountsfor the amount of background noise that is present in the sample.Background noise typically comprises low frequency emission produced,for example, by the intrinsic fluorescence of non-labeled particles thatare present in the sample, the buffer or diluent used in preparing thesample for analysis, Raman scattering and electronic noise. In someembodiments, the value assigned to the background noise is calculated asthe average background signal noise detected in a plurality of bins,which are measurements of photon signals that are detected in aninterrogation space during a predetermined length of time. Thus in someembodiments, background noise is calculated for each sample as a numberspecific to that sample.

Given the value for the background noise, the threshold energy level canbe assigned. As discussed above, the threshold value is determined todiscriminate true signals (due to fluorescence of a label) from thebackground noise. Care must be taken in choosing a threshold value suchthat the number of false positive signals from random noise is minimizedwhile the number of true signals which are rejected is also minimized.Methods for choosing a threshold value include determining a fixed valueabove the noise level and calculating a threshold value based on thedistribution of the noise signal. In one embodiment, the threshold isset at a fixed number of standard deviations above the background level.Assuming a Poisson distribution of the noise, using this method one canestimate the number of false positive signals over the time course ofthe experiment. In some embodiments, the threshold level is calculatedas a value of 4 sigma above the background noise. For example, given anaverage background noise level of 200 photons, the analyzer systemestablishes a threshold level of 4√200 above the averagebackground/noise level of 200 photons to be 256 photons. Thus, in someembodiments, determining the concentration of a label in a sampleincludes establishing the threshold level above which photon signalsrepresent the presence of a label. Conversely, photon signals that havean energy level that is not greater than that of the threshold levelindicate the absence of a label.

Many bin measurements are taken to determine the concentration of asample, and the absence or presence of a label is ascertained for eachbin measurement. Typically, 60,000 measurements or more can made in oneminute (e.g., in embodiments in which the bin size is 1 ms—for smallerbin sizes the number of measurements is correspondingly larger, e.g.,6,000,000 measurements per minute for a bin size of 10 microseconds).Thus, no single measurement is crucial and the method provides for ahigh margin of error. The bins that are determined not to contain alabel (“no” bins) are discounted and only the measurements made in thebins that are determined to contain label (“yes” bins) are accounted indetermining the concentration of the label in the processing sample.Discounting measurements made in the “no” bins or bins that are devoidof label increases the signal to noise ratio and the accuracy of themeasurements. Thus, in some embodiments, determining the concentrationof a label in a sample comprises detecting the bin measurements thatreflect the presence of a label.

The signal to noise ratio or the sensitivity of the analyzer system canbe increased by minimizing the time that background noise is detectedduring a bin measurement in which a particle-label complex is detected.For example, in a bin measurement lasting 1 millisecond during which oneparticle-label complex is detected when passing across an interrogationspace within 250 microseconds, 750 microseconds of the 1 millisecond arespent detecting background noise emission. The signal to noise ratio canbe improved by decreasing the bin time. In some embodiments, the bintime is 1 millisecond. In other embodiments, the bin time is 750, 500,250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds or10 microseconds. Other bin times are as described herein.

Other factors that affect measurements are the brightness or dimness ofthe fluorescent moiety, the flow rate, and the power of the laser.Various combinations of the relevant factors that allow for detection oflabel will be apparent to those of skill in the art. In someembodiments, the bin time is adjusted without changing the flow rate. Itwill be appreciated by those of skill in the art that as bin timedecreases, laser power output directed at the interrogation space mustincrease to maintain a constant total energy applied to theinterrogation space during the bin time. For example, if bin time isdecreased from 1000 microseconds to 250 microseconds, as a firstapproximation, laser power output must be increased approximatelyfour-fold. These settings allow for the detection of the same number ofphotons in a 250 μs as the number of photons counted during the 1000 μsgiven the previous settings, and allow for faster analysis of samplewith lower backgrounds and thus greater sensitivity. In addition, flowrates may be adjusted in order to speed processing of sample. Thesenumbers are merely exemplary, and the skilled practitioner can adjustthe parameters as necessary to achieve the desired result.

In some embodiments, the interrogation space encompasses the entirecross-section of the sample stream. When the interrogation spaceencompasses the entire cross-section of the sample stream, only thenumber of labels counted and the volume passing through a cross-sectionof the sample stream in a set length of time are needed to calculate theconcentration of the label in the processing sample. In someembodiments, the interrogation space can be defined to be smaller thanthe cross-sectional area of sample stream by, for example, theinterrogation space is defined by the size of the spot illuminated bythe laser beam. In some embodiments, the interrogation space can bedefined by adjusting the apertures 306 (FIG. 1A) or 358 and 359 (FIG.1B) of the analyzer and reducing the illuminated volume that is imagedby the objective lens to the detector. In the embodiments when theinterrogation space is defined to be smaller than the cross-sectionalarea of sample stream, the concentration of the label can be determinedby interpolation of the signal emitted by the complex from a standardcurve that is generated using one or more samples of known standardconcentrations. In yet other embodiments, the concentration of the labelcan be determined by comparing the measured particles to an internallabel standard. In embodiments when a diluted sample is analyzed, thedilution factor is accounted in calculating the concentration of themolecule of interest in the starting sample.

As discussed above, when the interrogation space encompasses the entirecross-section of the sample stream, only the number of labels countedpassing through a cross-section of the sample stream in a set length oftime (bin) and the volume of sample that was interrogated in the bin areneeded to calculate the concentration the sample. The total number oflabels contained in the “yes” bins is determined and related to thesample volume represented by the total number of bins used in theanalysis to determine the concentration of labels in the processingsample. Thus, in one embodiment, determining the concentration of alabel in a processing sample comprises determining the total number oflabels detected “yes” bins and relating the total number of detectedlabels to the total sample volume that was analyzed. The total samplevolume that is analyzed is the sample volume that is passed through thecapillary flow cell and across the interrogation space in a specifiedtime interval. Alternatively, the concentration of the label complex ina sample is determined by interpolation of the signal emitted by thelabel in a number of bins from a standard curve that is generated bydetermining the signal emitted by labels in the same number of bins bystandard samples containing known concentrations of the label.

In some embodiments, the number of individual labels that are detectedin a bin is related to the relative concentration of the particle in theprocessing sample. At relatively low concentrations, for example atconcentrations below about 10⁻¹⁶ M the number of labels is proportionalto the photon signal that is detected in a bin. Thus, at lowconcentrations of label the photon signal is provided as a digitalsignal. At relatively higher concentrations, for example atconcentrations greater than about 10⁻¹⁶ M, the proportionality of photonsignal to a label is lost as the likelihood of two or more labelscrossing the interrogation space at about the same time and beingcounted as one becomes significant. Thus, in some embodiments,individual particles in a sample of a concentration greater than about10⁻¹⁶ M are resolved by decreasing the length of time of the binmeasurement.

Alternatively, in other embodiments, the total photon signal that isemitted by a plurality of particles that are present in any one bin isdetected. These embodiments allow for single molecule detectors of theinvention wherein the dynamic range is at least 3, 3.5, 4, 4.5, 5.5, 6,6.5, 7, 7.5, 8, or more than 8 logs.

“Dynamic range,” as that term is used herein, refers to the range ofsample concentrations that may be quantitated by the instrument withoutneed for dilution or other treatment to alter the concentration ofsuccessive samples of differing concentrations, where concentrations aredetermined with an accuracy appropriate for the intended use. Forexample, if a microtiter plate contains a sample of 1 femtomolarconcentration for an analyte of interest in one well, a sample of 10,000femtomolar concentration for an analyte of interest in another well, anda sample of 100 femtomolar concentration for the analyte in a thirdwell, an instrument with a dynamic range of at least 4 logs and a lowerlimit of quantitation of 1 femtomolar is able to accurately quantitatethe concentration of all the samples without the need for furthertreatment to adjust concentration, e.g., dilution. Accuracy may bedetermined by standard methods, e.g., using a series of standards ofconcentrations that span the dynamic range and constructing a standardcurve. Standard measures of fit of the resulting standard curve may beused as a measure of accuracy, e.g., an r² greater than about 0.7, 0.75,0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

Increased dynamic range is achieved by altering the manner in which datafrom the detector is analyzed, and/or by the use of an attenuatorbetween the detector and the interrogation space. At the low end of therange, where processing sample is sufficiently dilute that eachdetection event, i.e., each burst of photons above a threshold level ina bin (the “event photons”), likely represents only one label, the datais analyzed to count detection events as single molecules. Thereby eachbin is analyzed as a simple “yes” or “no” for the presence of label, asdescribed above. For a more concentrated processing sample, where thelikelihood of two or more labels occupying a single bin becomessignificant, the number of event photons in a significant number of binsis found to be substantially greater than the number expected for asingle label, e.g., the number of event photons in a significant numberof bins corresponds to two-fold, three-fold, or more, than the number ofevent photons expected for a single label. For these samples, theinstrument changes its method of data analysis to one of integrating thetotal number of event photons for the bins of the processing sample.This total will be proportional to the total number of labels that werein all the bins. For an even more concentrated processing sample, wheremany labels are present in most bins, background noise becomes aninsignificant portion of the total signal from each bin, and theinstrument changes its method of data analysis to one of counting totalphotons per bin (including background). An even further increase indynamic range can be achieved by the use of an attenuator between theflow cell and the detector, when concentrations are such that theintensity of light reaching the detector would otherwise exceed thecapacity of the detector for accurately counting photons, i.e., saturatethe detector.

The instrument may include a data analysis system that receives inputfrom the detector and determines the appropriate analysis method for thesample being run, and outputs values based on such analysis. The dataanalysis system may further output instructions to use or not use anattenuator, if an attenuator is included in the instrument.

By utilizing such methods, the dynamic range of the instrument can bedramatically increased. Thus, in some embodiments, the instrument iscapable of measuring concentrations of samples over a dynamic range ofmore than about 1000 (3 log), 10,000 (4 log), 100,000 (5 log), 350,000(5.5 log), 1,000,000 (6 log), 3,500,000 (6.5 log), 10,000,000 (7 log),35,000,000 (7.5 log), or 100,000,000 (8 log). In some embodiments, theinstrument is capable of measuring concentrations of samples over adynamic range of more than about 100,000 (5 log). In some embodiments,the instrument is capable of measuring concentrations of samples over adynamic range of more than about 1,000,000 (6 log). In some embodiments,the instrument is capable of measuring concentrations of samples over adynamic range of more than about 10,000,000 (7 log). In someembodiments, the instrument is capable of measuring the concentrationsof samples over a dynamic range of from about 1-10 femtomolar to atleast about 1000; 10,000; 100,000; 350,000; 1,000,000; 3,500,000;10,000,000; or 35,000,000 femtomolar. In some embodiments, theinstrument is capable of measuring the concentrations of samples over adynamic range of from about 1-10 femtomolar to at least about 10,000femtomolar. In some embodiments, the instrument is capable of measuringthe concentrations of samples over a dynamic range of from about 1-10femtomolar to at least about 100,000 femtomolar. In some embodiments,the instrument is capable of measuring the concentrations of samplesover a dynamic range of from about 1-10 femtomolar to at least about1,000,000 femtomolar. In some embodiments, the instrument is capable ofmeasuring the concentrations of samples over a dynamic range of fromabout 1-10 femtomolar to at least about 10,000,000.

In some embodiments, an analyzer or analyzer system of the invention iscapable of detecting an analyte, e.g., a biomarker, at a limit ofdetection of less than 1 nanomolar, or 1 picomolar, or 1 femtomolar, or1 attomolar, or 1 zeptomolar. In some embodiments, the analyzer oranalyzer system is capable of detecting a change in concentration of theanalyte, or of multiple analytes, e.g., a biomarker or biomarkers, fromone sample to another sample of less than about 0.1%, 1%, 2%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70% or 80% when the biomarker is present at aconcentration of less than 1 nanomolar, or 1 picomolar, or 1 femtomolar,or 1 attomolar, or 1 zeptomolar, in the samples, and when the size ofeach of the sample is less than about 100, 50, 40, 30, 20, 10, 5, 2, 1,0.1, 0.01, 0.001, or 0.0001 μl. In some embodiments, the analyzer oranalyzer system is capable of detecting a change in concentration of theanalyte from a first sample to a second sample of less than about 20%,when the analyte is present at a concentration of less than about 1picomolar, and when the size of each of the samples is less than about50 μl. In some embodiments, the analyzer or analyzer system is capableof detecting a change in concentration of the analyte from a firstsample to a second sample of less than about 20%, when the analyte ispresent at a concentration of less than about 100 femtomolar, and whenthe size of each of the samples is less than about 50 μl. In someembodiments, the analyzer or analyzer system is capable of detecting achange in concentration of the analyte from a first sample to a secondsample of less than about 20%, when the analyte is present at aconcentration of less than about 50 femtomolar, and when the size ofeach of the samples is less than about 50 μl. In some embodiments, theanalyzer or analyzer system is capable of detecting a change inconcentration of the analyte from a first sample to a second sample ofless than about 20%, when the analyte is present at a concentration ofless than about 5 femtomolar, and when the size of each of the samplesis less than about 50 μl. In some embodiments, the analyzer or analyzersystem is capable of detecting a change in concentration of the analytefrom a first sample to a second sample of less than about 20%, when theanalyte is present at a concentration of less than about 5 femtomolar,and when the size of each of the samples is less than about 5 μl. Insome embodiments, the analyzer or analyzer system is capable ofdetecting a change in concentration of the analyte from a first sampleto a second sample of less than about 20%, when the analyte is presentat a concentration of less than about 1 femtomolar, and when the size ofeach of the samples is less than about 5 μl.

The single molecule detectors of the present invention are capable ofdetecting molecules of interest in a highly sensitive manner with a verylow coefficient of variation (CV). In some embodiments, the CV is lessthan about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, or less than about 1%.In some embodiments, the CV is less than about 50%. In some embodiments,the CV is less than about 40%. In some embodiments, the CV is less thanabout 30%. In some embodiments, the CV is less than about 25%. In someembodiments, the CV is less than about 20%. In some embodiments, the CVis less than about 15%. In some embodiments, the CV is less than about10%. In some embodiments, the CV is less than about 5%. In someembodiments, the CV is less than about 1%. In some embodiments, thelimit of detection (LOD) is less than about 100 pg/ml and the CV is lessthan about 10%. In some embodiments, the limit of detection (LOD) isless than about 50 pg/ml and the CV is less than about 10%. In someembodiments, the limit of detection (LOD) is less than about 40 pg/mland the CV is less than about 10%. In some embodiments, the limit ofdetection (LOD) is less than about 30 pg/ml and the CV is less thanabout 10%. In some embodiments, the limit of detection (LOD) is lessthan about 20 pg/ml and the CV is less than about 10%. In someembodiments, the limit of detection (LOD) is less than about 15 pg/mland the CV is less than about 10%. In some embodiments, the limit ofdetection (LOD) is less than about 10 pg/ml and the CV is less thanabout 10%. In some embodiments, the limit of detection (LOD) is lessthan about 5 pg/ml and the CV is less than about 10%. In someembodiments, the limit of detection (LOD) is less than about 1 pg/ml andthe CV is less than about 10%. In some embodiments, the limit ofdetection (LOD) is less than about 0.05 pg/ml and the CV is less thanabout 10%. In some embodiments, the limit of detection (LOD) is lessthan about 0.01 pg/ml and the CV is less than about 10%. In someembodiments, the limit of detection (LOD) is less than about 10 pg/mland the CV is less than about 50%. In some embodiments, the limit ofdetection (LOD) is less than about 10 pg/ml and the CV is less thanabout 25%. In some embodiments, the limit of detection (LOD) is lessthan about 10 pg/ml and the CV is less than about 10%. In someembodiments, the limit of detection (LOD) is less than about 10 pg/mland the CV is less than about 5%. In some embodiments, the limit ofdetection (LOD) is less than about 10 pg/ml and the CV is less thanabout 1%. In some embodiments, the limit of detection (LOD) is less thanabout 5 pg/ml and the CV is less than about 100%. In some embodiments,the limit of detection (LOD) is less than about 5 pg/ml and the CV isless than about 50%. In some embodiments, the limit of detection (LOD)is less than about 5 pg/ml and the CV is less than about 25%. In someembodiments, the limit of detection (LOD) is less than about 5 pg/ml andthe CV is less than about 10%. In some embodiments, the limit ofdetection (LOD) is less than about 5 pg/ml and the CV is less than about5%. In some embodiments, the limit of detection (LOD) is less than about5 pg/ml and the CV is less than about 1%. In some embodiments, the limitof detection (LOD) is less than about 1 pg/ml and the CV is less thanabout 100%. In some embodiments, the limit of detection (LOD) is lessthan about 1 pg/ml and the CV is less than about 50%. In someembodiments, the limit of detection (LOD) is less than about 1 pg/ml andthe CV is less than about 25%. In some embodiments, the limit ofdetection (LOD) is less than about 1 pg/ml and the CV is less than about10%. In some embodiments, the limit of detection (LOD) is less thanabout 1 pg/ml and the CV is less than about 5%. In some embodiments, thelimit of detection (LOD) is less than about 1 pg/ml and the CV is lessthan about 1%.

V. INSTRUMENTS AND SYSTEMS SUITABLE FOR HIGHLY SENSITIVE ANALYSIS OFMOLECULES

The methods of the invention utilize analytical instruments of highsensitivity, e.g., single molecule detectors. Such single moleculedetectors include embodiments as hereinafter described.

In some embodiments, the invention provides an analyzer system kit fordetecting a single protein molecule in a sample, said system includes ananalyzer system for detecting a single protein molecule in a sample andleast one label that includes a fluorescent moiety and a binding partnerfor the protein molecule, where the analyzer includes an electromagneticradiation source for stimulating the fluorescent moiety; a capillaryflow cell for passing the label; a source of motive force for moving thelabel in the capillary flow cell; an interrogation space defined withinthe capillary flow cell for receiving electromagnetic radiation emittedfrom the electromagnetic source; and an electromagnetic radiationdetector operably connected to the interrogation space for measuring anelectromagnetic characteristic of the stimulated fluorescent moiety,where the fluorescent moiety is capable of emitting at least about 200photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot not lessthan about 5 microns in diameter that contains the moiety, and where thetotal energy directed at the spot by the laser is no more than about 3microJoules.

One embodiment of an analyzer kit of the invention is depicted in FIG.15. The kit includes a label for a protein molecule that includes abinding partner for a protein molecule and a fluorescent moiety. The kitfurther includes an analyzer system for detecting a single proteinmolecule (300) that includes an electromagnetic radiation source 301 forstimulating the fluorescent moiety, a capillary flow cell 313 forpassing the label; a source of motive force for moving the label in thecapillary flow cell (not shown); an interrogation space defined withinthe capillary flow cell for receiving electromagnetic radiation emittedfrom the electromagnetic source 314 (FIG. 2A); and an electromagneticradiation detector 309 operably connected to the interrogation space formeasuring an electromagnetic characteristic of the stimulatedfluorescent moiety, where the fluorescent moiety is capable of emittingat least about 200 photons when simulated by a laser emitting light atthe excitation wavelength of the moiety, where the laser is focused on aspot not less than about 5 microns in diameter that contains the moiety,and where the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the beam 311 from anelectromagnetic radiation source 301 is focused by the microscopeobjective 315 to form one interrogation space 314 (FIG. 2A) within thecapillary flow cell 313. The microscope objective may have a numericalaperture of equal to or greater than 0.7, 0.8, 0.9, or 1.0 in someembodiments.

In some embodiments of the analyzer system kit, the analyzer comprisesnot more than one interrogation space. In some embodiments, theelectromagnetic radiation source is a laser that has a power output ofat least about 3, 5, 10, or 20 mW. In some embodiments, the fluorescentmoiety comprises a fluorescent molecule. In some embodiments, thefluorescent molecule is a dye molecule, such as a dye molecule thatcomprises at least one substituted indolium ring system in which thesubstituent on the 3-carbon of the indolium ring contains a chemicallyreactive group or a conjugated substance. In some embodiments, thefluorescent moiety is a quantum dot. In some embodiments, theelectromagnetic radiation source is a continuous wave electromagneticradiation source, such as a light-emitting diode or a continuous wavelaser. In some embodiments, the motive force is pressure. In someembodiments, the detector is an avalanche photodiode detector. In someembodiments, the analyzer utilizes a confocal optical arrangement fordeflecting a laser beam onto said interrogation space and for imagingsaid stimulated dye molecule (shown in FIGS. 1, 3), wherein saidconfocal optical arrangement comprises an objective lens having anumerical aperture of at least about 0.8. In some embodiments, theanalyzer further comprises a sampling system capable of automaticallysampling a plurality of samples and providing a fluid communicationbetween a sample container and said interrogation space. In someembodiments, the analyzer system further comprises a sample recoverysystem in fluid communication with said interrogation space, whereinsaid recovery system is capable of recovering substantially all of saidsample. In some embodiments, the kit further includes instructions foruse of the system.

A. Apparatus/System

In one aspect, the methods described herein utilize an analyzer systemcapable of detecting a single molecule in a sample. In one embodiment,the analyzer system is capable of single molecule detection of afluorescently labeled particle wherein the analyzer system detectsenergy emitted by an excited fluorescent label in response to exposureby an electromagnetic radiation source when the single particle ispresent in an interrogation space defined within a capillary flow cellfluidly connected to the sampling system of the analyzer system. In afurther embodiment of the analyzer system, the single particle movesthrough the interrogation space of the capillary flow cell by means of amotive force. In another embodiment of the analyzer system, an automaticsampling system may be included in the analyzer system for introducingthe sample into the analyzer system. In another embodiment of theanalyzer system, a sample preparation system may be included in theanalyzer system for preparing a sample. In a further embodiment, theanalyzer system may contain a sample recovery system for recovering atleast a portion of the sample after analysis is complete.

In one aspect, the analyzer system consists of an electromagneticradiation source for exciting a single particle labeled with afluorescent label. In one embodiment, the electromagnetic radiationsource of the analyzer system is a laser. In a further embodiment, theelectromagnetic radiation source is a continuous wave laser.

In a typical embodiment, the electromagnetic radiation source excites afluorescent moiety attached to a label as the label passes through theinterrogation space of the capillary flow cell. In some embodiments, thefluorescent label moiety includes one or more fluorescent dye molecules.In some embodiments, the fluorescent label moiety is a quantum dot. Anyfluorescent moiety as described herein may be used in the label.

A label is exposed to electromagnetic radiation when the label passesthrough an interrogation space located within the capillary flow cell.The interrogation space is typically fluidly connected to a samplingsystem. In some embodiments the label passes through the interrogationspace of the capillary flow cell due to a motive force to advance thelabel through the analyzer system. The interrogation space is positionedsuch that it receives electromagnetic radiation emitted from theradiation source. In some embodiments, the sampling system is anautomated sampling system capable of sampling a plurality of sampleswithout intervention from a human operator.

The label passes through the interrogation space and emits a detectableamount of energy when excited by the electromagnetic radiation source.In one embodiment, an electromagnetic radiation detector is operablyconnected to the interrogation space. The electromagnetic radiationdetector is capable of detecting the energy emitted by the label, e.g.,by the fluorescent moiety of the label.

In a further embodiment of the analyzer system, the system furtherincludes a sample preparation mechanism where a sample may be partiallyor completely prepared for analysis by the analyzer system. In someembodiments of the analyzer system, the sample is discarded after it isanalyzed by the system. In other embodiments, the analyzer systemfurther includes a sample recovery mechanism whereby at least a portion,or alternatively all or substantially all, of the sample may berecovered after analysis. In such an embodiment, the sample can bereturned to the origin of the sample. In some embodiments, the samplecan be returned to microtiter wells on a sample microtiter plate. Theanalyzer system typically further consists of a data acquisition systemfor collecting and reporting the detected signal.

B. Single Particle Analyzer

As shown in FIG. 1A, described herein is one embodiment of an analyzersystem 300. The analyzer system 300 includes an electromagneticradiation source 301, a mirror 302, a lens 303, a capillary flow cell313, a microscopic objective lens 305, an aperture 306, a detector lens307, a detector filter 308, a single photon detector 309, and aprocessor 310 operatively connected to the detector.

In operation the electromagnetic radiation source 301 is aligned so thatits output 311 is reflected off of a front surface 312 of mirror 302.The lens 303 focuses the beam 311 onto a single interrogation space (anillustrative example of an interrogation space 314 is shown in FIG. 2A)in the capillary flow cell 313. The microscope objective lens 305collects light from sample particles and forms images of the beam ontothe aperture 306. The aperture 306 affects the fraction of light emittedby the specimen in the interrogation space of the capillary flow cell313 that can be collected. The detector lens 307 collects the lightpassing through the aperture 306 and focuses the light onto an activearea of the detector 309 after it passes through the detector filters308. The detector filters 308 minimize aberrant noise signals due tolight scatter or ambient light while maximizing the signal emitted bythe excited fluorescent moiety bound to the particle. The processor 310processes the light signal from the particle according to the methodsdescribed herein.

In one embodiment, the microscope objective lens 305 is a high numericalaperture microscope objective. As used herein, “high numerical aperturelens” include a lens with a numerical aperture of equal to or greaterthan 0.6. The numerical aperture is a measure of the number of highlydiffracted image-forming light rays captured by the objective. A highernumerical aperture allows increasingly oblique rays to enter theobjective lens and thereby produce a more highly resolved image.Additionally, the brightness of an image increases with a highernumerical aperture. High numerical aperture lenses are commerciallyavailable from a variety of vendors, and any one lens having a numericalaperture of equal to or greater than approximately 0.6 may be used inthe analyzer system. In some embodiments, the lens has a numericalaperture of about 0.6 to about 1.3. In some embodiments, the lens has anumerical aperture of about 0.6 to about 1.0. In some embodiments, thelens has a numerical aperture of about 0.7 to about 1.2. In someembodiments, the lens has a numerical aperture of about 0.7 to about1.0. In some embodiments, the lens has a numerical aperture of about 0.7to about 0.9. In some embodiments, the lens has a numerical aperture ofabout 0.8 to about 1.3. In some embodiments, the lens has a numericalaperture of about 0.8 to about 1.2. In some embodiments, the lens has anumerical aperture of about 0.8 to about 1.0. In some embodiments, thelens has a numerical aperture of at least about 0.6. In someembodiments, the lens has a numerical aperture of at least about 0.7. Insome embodiments, the lens has a numerical aperture of at least about0.8. In some embodiments, the lens has a numerical aperture of at leastabout 0.9. In some embodiments, the lens has a numerical aperture of atleast about 1.0. In some embodiments, the aperture of the microscopeobjective lens 305 is approximately 1.25. In an embodiment where amicroscope objective lens 305 of 0.8 is used, a Nikon 60X/0.8 NAAchromat lens (Nikon, Inc., USA) can be used.

In some embodiments, the electromagnetic radiation source 301 is a laserthat emits light in the visible spectrum. In all embodiments, theelectromagnetic radiation source is set such that wavelength of thelaser is set such that it is of a sufficient wavelength to excite thefluorescent label attached to the particle. In some embodiments, thelaser is a continuous wave laser with a wavelength of 639 nm. In otherembodiments, the laser is a continuous wave laser with a wavelength of532 nm. In other embodiments, the laser is a continuous wave laser witha wavelength of 422 nm. In other embodiments, the laser is a continuouswave laser with a wavelength of 405 nm. Any continuous wave laser with awavelength suitable for exciting a fluorescent moiety as used in themethods and compositions of the invention may be used without departingfrom the scope of the invention.

In a single particle analyzer system 300, as each particle passesthrough the beam 311 of the electromagnetic radiation source, theparticle enters into an excited state. When the particle relaxes fromits excited state, a detectable burst of light is emitted. Theexcitation-emission cycle is repeated many times by each particle in thelength of time it takes for it to pass through the beam allowing theanalyzer system 300 to detect tens to thousands of photons for eachparticle as it passes through an interrogation space 314. Photonsemitted by fluorescent particles are registered by the detector 309(FIG. 1A) with a time delay indicative of the time for the particlelabel complex to pass through the interrogation space. The photonintensity is recorded by the detector 309 and sampling time is dividedinto bins, which are uniform, arbitrary, time segments with freelyselectable time channel widths. The number of signals contained in eachbin evaluated. One or a combination of several statistical analyticalmethods are employed in order to determine when a particle is present.Such methods include determining the baseline noise of the analyzersystem and setting a signal strength for the fluorescent label at astatistical level above baseline noise to eliminate false positivesignals from the detector.

The electromagnetic radiation source 301 is focused onto a capillaryflow cell 313 of the analyzer system 300 where the capillary flow cell313 is fluidly connected to the sample system. An interrogation space314 is shown in FIG. 2A. The beam 311 from the continuous waveelectromagnetic radiation source 301 of FIG. 1A is optically focused toa specified depth within the capillary flow cell 313. The beam 311 isdirected toward the sample-filled capillary flow cell 313 at an angleperpendicular to the capillary flow cell 313. The beam 311 is operatedat a predetermined wavelength that is selected to excite a particularfluorescent label used to label the particle of interest. The size orvolume of the interrogation space 314 is determined by the diameter ofthe beam 311 together with the depth at which the beam 311 is focused.Alternatively, the interrogation space can be determined by running acalibration sample of known concentration through the analyzer system.

When single molecules are detected in the sample concentration, the beamsize and the depth of focus required for single molecule detection areset and thereby define the size of the interrogation space 314. Theinterrogation space 314 is set such that, with an appropriate sampleconcentration, only one particle is present in the interrogation space314 during each time interval over which time observations are made.

It will be appreciated that the detection interrogation volume asdefined by the beam is not perfectly spherically shaped, and typicallyis a “bow-tie” shape. However, for the purposes of definition, “volumes”of interrogation spaces are defined herein as the volume encompassed bya sphere of a diameter equal to the focused spot diameter of the beam.The focused spot of the beam 311 may have various diameters withoutdeparting from the scope of the present invention. In some embodiments,the diameter of the focused spot of the beam is about 1 to about 5, 10,15, or 20 microns, or about 5 to about 10, 15, or 20 microns, or about10 to about 20 microns, or about 10 to about 15 microns. In someembodiments, the diameter of the focused spot of the beam is about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20microns. In some embodiments, the diameter of the focused spot of thebeam is about 5 microns. In some embodiments, the diameter of thefocused spot of the beam is about 10 microns. In some embodiments, thediameter of the focused spot of the beam is about 12 microns. In someembodiments, the diameter of the focused spot of the beam is about 13microns. In some embodiments, the diameter of the focused spot of thebeam is about 14 microns. In some embodiments, the diameter of thefocused spot of the beam is about 15 microns. In some embodiments, thediameter of the focused spot of the beam is about 16 microns. In someembodiments, the diameter of the focused spot of the beam is about 17microns. In some embodiments, the diameter of the focused spot of thebeam is about 18 microns. In some embodiments, the diameter of thefocused spot of the beam is about 19 microns. In some embodiments, thediameter of the focused spot of the beam is about 20 microns.

In an alternate embodiment of the single particle analyzer system, morethan one electromagnetic radiation source can be used to exciteparticles labeled with fluorescent labels of different wavelengths. Inanother alternate embodiment, more than one interrogation space in thecapillary flow cell can be used. In another alternate embodiment,multiple detectors can be employed to detect different emissionwavelengths from the fluorescent labels. An illustration incorporatingeach of these alternative embodiments of an analyzer system is shown inFIG. 1B. These embodiments are incorporated by reference from previousU.S. patent application Ser. No. 11/048,660.

In some embodiments of the analyzer system 300, a motive force isrequired to move a particle through the capillary flow cell 313 of theanalyzer system 300. In one embodiment, the motive force can be a formof pressure. The pressure used to move a particle through the capillaryflow cell can be generated by a pump. In some embodiments, a Scivex,Inc. HPLC pump can be used. In some embodiments where a pump is used asa motive force, the sample can pass through the capillary flow cell at arate of 1 μL/min to about 20 μL/min, or about 5 μL/min to about 20μL/min. In some embodiments, the sample can pass through the capillaryflow cell at a rate of about 5 μL/min. In some embodiments, the samplecan pass through the capillary flow cell at a rate of about 10 μL/min.In some embodiments, the sample can pass through the capillary flow cellat a rate of about 15 μL/min. In some embodiments, the sample can passthrough the capillary flow cell at a rate of about 20 μL/min. In someembodiments, an electrokinetic force can be used to move the particlethrough the analyzer system. Such a method has been previously disclosedand is incorporated by reference from previous U.S. patent applicationSer. No. 11/048,660.

In one aspect of the analyzer system 300, the detector 309 of theanalyzer system detects the photons emitted by the fluorescent label. Inone embodiment, the photon detector is a photodiode. In a furtherembodiment, the detector is an avalanche photodiode detector. In someembodiments, the photodiodes can be silicon photodiodes with awavelength detection of 190 nm and 1100 nm. When germanium photodiodesare used, the wavelength of light detected is between 400 nm to 1700 nm.In other embodiments, when an indium gallium arsenide photodiode isused, the wavelength of light detected by the photodiode is between 800nm and 2600 nm. When lead sulfide photodiodes are used as detectors, thewavelength of light detected is between 1000 nm and 3500 nm.

In some embodiments, the optics of the electromagnetic radiation source301 and the optics of the detector 309 are arranged in a conventionaloptical arrangement. In such an arrangement, the electromagneticradiation source and the detector are aligned on different focal planes.The arrangement of the laser and the detector optics of the analyzersystem as shown in FIGS. 1A and 1B is that of a conventional opticalarrangement.

In some embodiments, the optics of the electromagnetic radiation sourceand the optics of the detector are arranged in a confocal opticalarrangement. In such an arrangement, the electromagnetic radiationsource 301 and the detector 309 are aligned on the same focal plane. Theconfocal arrangement renders the analyzer more robust because theelectromagnetic radiation source 301 and the detector optics 309 do notneed to be realigned if the analyzer system is moved. This arrangementalso makes the use of the analyzer more simplified because it eliminatesthe need to realign the components of the analyzer system. The confocalarrangement for the analyzer 300 (FIG. 1A) and the analyzer 355 (FIG.1B) are shown in FIGS. 3A and 3B respectively. FIG. 3A shows that thebeam 311 from an electromagnetic radiation source 301 is focused by themicroscope objective 315 to form one interrogation space 314 (FIG. 2A)within the capillary flow cell 313. A dichroic mirror 316, whichreflects laser light but passes fluorescent light, is used to separatethe fluorescent light from the laser light. Filter 317 that ispositioned in front of the detector eliminates any non-fluorescent lightat the detector. In some embodiments, an analyzer system configured in aconfocal arrangement can comprise two or more interrogations spaces.Such a method has been previously disclosed and is incorporated byreference from previous U.S. patent application Ser. No. 11/048,660.

The laser can be a tunable dye laser, such as a helium-neon laser. Thelaser can be set to emit a wavelength of 632.8 nm. Alternatively, thewavelength of the laser can be set to emit a wavelength of 543.5 nm or1523 nm. Alternatively, the electromagnetic laser can be an argon ionlaser. In such an embodiment, the argon ion laser can be operated as acontinuous gas laser at about 25 different wavelengths in the visiblespectrum, the wavelength set between 408.9 and 686.1 nm but at itsoptimum performance set between 488 and 514.5 nm.

1. Electromagnetic Radiation Source

In some embodiments of the analyzer system a chemiluminescent label maybe used. In such an embodiment, it may not be necessary to utilize an EMsource for detection of the particle. In another embodiment, theextrinsic label or intrinsic characteristic of the particle is alight-interacting label or characteristic, such as a fluorescent labelor a light-scattering label. In such an embodiment, a source of EMradiation is used to illuminate the label and/or the particle. EMradiation sources for excitation of fluorescent labels are preferred.

In some embodiments, the analyzer system consists of an electromagneticradiation source 301. Any number of radiation sources may be used in anyone analyzer system 300 without departing from the scope of theinvention. Multiple sources of electromagnetic radiation have beenpreviously disclosed and are incorporated by reference from previousU.S. patent application Ser. No. 11/048,660. In some embodiments, allthe continuous wave electromagnetic (EM) radiation sources emitelectromagnetic radiation at the same wavelengths. In other embodiments,different sources emit different wavelengths of EM radiation.

In one embodiment, the EM source(s) 301, 351, 352 are continuous wavelasers producing wavelengths of between 200 nm and 1000 nm. Such EMsources have the advantage of being small, durable and relativelyinexpensive. In addition, they generally have the capacity to generatelarger fluorescent signals than other light sources. Specific examplesof suitable continuous wave EM sources include, but are not limited to:lasers of the argon, krypton, helium-neon, helium-cadmium types, as wellas, tunable diode lasers (red to infrared regions), each with thepossibility of frequency doubling. The lasers provide continuousillumination with no accessory electronic or mechanical devices, such asshutters, to interrupt their illumination. In an embodiment where acontinuous wave laser is used, an electromagnetic radiation source of 3mW may be of sufficient energy to excite a fluorescent label. A beamfrom a continuous wave laser of such energy output may be between 2 to 5μm in diameter. The time of exposure of the particle to laser beam inorder to be exposed to 3 mW may be a time period of about 1 msec. Inalternate embodiments, the time of exposure to the laser beam may beequal to or less than about 500 μsec. In an alternate embodiment, thetime of exposure may be equal to or less than about 100 μsec. In analternate embodiment, the time of exposure may be equal to or less thanabout 50 μsec. In an alternate embodiment, the time of exposure may beequal to or less than about 10 μsec.

LEDs are another low-cost, high reliability illumination source. Recentadvances in ultra-bright LEDs and dyes with high absorptioncross-section and quantum yield support the applicability of LEDs tosingle particle detection. Such lasers could be used alone or incombination with other light sources such as mercury arc lamps,elemental arc lamps, halogen lamps, arc discharges, plasma discharges,light-emitting diodes, or combination of these.

In other embodiments, the EM source could be in the form of a pulse wavelaser. In such an embodiment, the pulse size of the laser is animportant factor. In such an embodiment, the size, focus spot, and thetotal energy emitted by the laser is important and must be of sufficientenergy as to be able to excite the fluorescent label. When a pulse laseris used, a pulse of longer duration may be required. In some embodimentsa laser pulse of 2 nanoseconds may be used. In some embodiments a laserpulse of 5 nanoseconds may be used. In some embodiments a pulse ofbetween 2 to 5 nanoseconds may be used.

The optimal laser intensity depends on the photo bleachingcharacteristics of the single dyes and the length of time required totraverse the interrogation space (including the speed of the particle,the distance between interrogation spaces if more than one is used andthe size of the interrogation space(s)). To obtain a maximal signal, itis desirable to illuminate the sample at the highest intensity whichwill not result in photo bleaching a high percentage of the dyes. Thepreferred intensity is one such that no more that 5% of the dyes arebleached by the time the particle has traversed the interrogation space.

The power of the laser is set depending on the type of dye moleculesthat need to be stimulated and the length of time the dye molecules arestimulated, and/or the speed with which the dye molecules pass throughthe capillary flow cell. Laser power is defined as the rate at whichenergy is delivered by the beam and is measured in units ofJoules/second, or Watts. It will be appreciated that the greater thepower output of the laser, the shorter the time that the laserilluminates the particle may be, while providing a constant amount ofenergy to the interrogation space while the particle is passing throughthe space. Thus, in some embodiments, the combination of laser power andtime of illumination is such that the total energy received by theinterrogation space during the time of illumination is more than about0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, or 100 microJoule. In some embodiments, thecombination of laser power and time of illumination is such that thetotal energy received by the interrogation space during the time ofillumination is less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 110 microJoule.In some embodiments, the combination of laser power and time ofillumination is such that the total energy received by the interrogationspace during the time of illumination is between about 0.1 and 100microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 1and 100 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 1and 50 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 2and 50 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 60 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 50 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 40 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 30 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is about 1microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 3microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 5microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 10microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 15microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 20microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 30microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 40microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 50microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 60microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 70microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 80microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 90microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 100microJoule.

In some embodiments, the laser power output is set to at least about 1mW, 2 mW, 3 mW, 4 mW, 5 mW, 6, mw, 7 mW, 8 mW, 9 mW, 10 mW, 13 mW, 15mW, 20 mW, 25 mW, 30 mW, 40 mW, 50 mW, 60 mW, 70 mW, 80 mW, 90 mW, 100mW, or more than 100 mW. In some embodiments, the laser power output isset to at least about 1 mW. In some embodiments, the laser power outputis set to at least about 3 mW. In some embodiments, the laser poweroutput is set to at least about 5 mW. In some embodiments, the laserpower output is set to at least about 10 mW. In some embodiments, thelaser power output is set to at least about 15 mW. In some embodiments,the laser power output is set to at least about 20 mW. In someembodiments, the laser power output is set to at least about 30 mW. Insome embodiments, the laser power output is set to at least about 40 mW.In some embodiments, the laser power output is set to at least about 50mW. In some embodiments, the laser power output is set to at least about60 mW. In some embodiments, the laser power output is set to at leastabout 90 mW.

The time that the laser illuminates the interrogation space can be setto no less than about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 150, 300, 350, 400, 450, 500, 600, 700, 800, 900, or1000 microseconds. The time that the laser illuminates the interrogationspace can be set to no more than about 2, 3, 4, 5, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 150, 300, 350, 400, 450, 500, 600,700, 800, 900, 1000, 1500, or 2000 microseconds. The time that the laserilluminates the interrogation space can be set between about 1 and 1000microseconds. The time that the laser illuminates the interrogationspace can be set between about 5 and 500 microseconds. The time that thelaser illuminates the interrogation space can be set between about 5 and100 microseconds. The time that the laser illuminates the interrogationspace can be set between about 10 and 100 microseconds. The time thatthe laser illuminates the interrogation space can be set between about10 and 50 microseconds. The time that the laser illuminates theinterrogation space can be set between about 10 and 20 microseconds. Thetime that the laser illuminates the interrogation space can be setbetween about 5 and 50 microseconds. The time that the laser illuminatesthe interrogation space can be set between about 1 and 100 microseconds.In some embodiments, the time that the laser illuminates theinterrogation space is about 1 microsecond. In some embodiments, thetime that the laser illuminates the interrogation space is about 5microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 10 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 25microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 50 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 100microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 250 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 500microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 1000 microseconds.

For example, the time that the laser illuminates the interrogation spacecan be set to 1 millisecond, 250 microseconds, 100 microseconds, 50microseconds, 25 microseconds or 10 microseconds with a laser thatprovides a power output of 3 mW, 4 mw, 5 mW, or more than 5 mW. In someembodiments, a label is illuminated with a laser that provides a poweroutput of 3 mW and illuminates the label for about 1000 microseconds. Inother embodiments, a label is illuminated for less than 1000milliseconds with a laser providing a power output of not more thanabout 20 mW. In other embodiments, the label is illuminated with a laserpower output of 20 mW for less than or equal to about 250 microseconds.In some embodiments, the label is illuminated with a laser power outputof about 5 mW for less than or equal to about 1000 microseconds.

2. Capillary Flow Cell

The capillary flow cell is fluidly connected to the sample system. Inone embodiment, the interrogation space 314 of an analyzer system isdetermined by the cross sectional area of the corresponding beam 311 andby a segment of the beam within the field of view of the detector 309.In one embodiment of the analyzer system, the interrogation space 314has a volume, as defined herein, of between about between about 0.01 and500 pL, or between about 0.01 pL and 100 pL, or between about 0.01 pLand 10 pL, or between about 0.01 pL and 1 pL, or between about 0.01 pLand 0.5 pL, or between about 0.02 pL and about 300 pL, or between about0.02 pL and about 50 pL or between about 0.02 pL and about 5 pL orbetween about 0.02 pL and about 0.5 pL or between about 0.02 pL andabout 2 pL, or between about 0.05 pL and about 50 pL, or between about0.05 pL and about 5 pL, or between about 0.05 pL and about 0.5 pL, orbetween about 0.05 pL and about 0.2 pL, or between about 0.1 pL andabout 25 pL. In some embodiments, the interrogation space has a volumebetween about 0.01 pL and 10 pL. In some embodiments, the interrogationspace 314 has a volume between about 0.01 pL and 1 pL. In someembodiments, the interrogation space 314 has a volume between about 0.02pL and about 5 pL. In some embodiments, the interrogation space 314 hasa volume between about 0.02 pL and about 0.5 pL. In some embodiments,the interrogation space 314 has a volume between about 0.05 pL and about0.2 pL. In some embodiments, the interrogation space 314 has a volume ofabout 0.1 pL. Other useful interrogation space volumes are as describedherein. It should be understood by one skilled in the art that theinterrogation space 314 can be selected for maximum performance of theanalyzer. Although very small interrogation spaces have been shown tominimize the background noise, large interrogation spaces have theadvantage that low concentration samples can be analyzed in a reasonableamount of time. In embodiments in which two interrogation spaces 370 and371 are used, volumes such as those described herein for a singleinterrogation space 314 may be used.

In one embodiment of the present invention, the interrogation spaces arelarge enough to allow for detection of particles at concentrationsranging from about 1000 femtomolar (fM) to about 1 zeptomolar (zM). Inone embodiment of the present invention, the interrogation spaces arelarge enough to allow for detection of particles at concentrationsranging from about 1000 fM to about 1 attomolar (aM). In one embodimentof the present invention, the interrogation spaces are large enough toallow for detection of particles at concentrations ranging from about 10fM to about 1 attomolar (aM). In many cases, the large interrogationspaces allow for the detection of particles at concentrations of lessthan about 1 fM without additional pre-concentration devices ortechniques. One skilled in the art will recognize that the mostappropriate interrogation space size depends on the brightness of theparticles to be detected, the level of background signal, and theconcentration of the sample to be analyzed.

The size of the interrogation space 314 can be limited by adjusting theoptics of the analyzer. In one embodiment, the diameter of the beam 311can be adjusted to vary the volume of the interrogation space 314. Inanother embodiment, the field of view of the detector 309 can be varied.Thus, the source 301 and the detector 309 can be adjusted so that singleparticles will be illuminated and detected within the interrogationspace 314. In another embodiment, the width of aperture 306 (FIG. 1A)that determine the field of view of the detector 309 is variable. Thisconfiguration allows for altering the interrogation space, in near realtime, to compensate for more or less concentrated samples, ensuring alow probability of two or more particles simultaneously being within aninterrogation space. Similar alterations for two or more interrogationspaces, 370 and 371, may performed.

In another embodiment, the interrogation space can be defined throughthe use of a calibration sample of known concentration that is passedthrough the capillary flow cell prior to the actual sample being tested.When only one single particle is detected at a time in the calibrationsample as the sample is passing through the capillary flow cell, thedepth of focus together with the diameter of the beam of theelectromagnetic radiation source determines the size of theinterrogation space in the capillary flow cell.

Physical constraints to the interrogation spaces can also be provided bya solid wall. In one embodiment, the wall is one or more of the walls ofa flow cell 313 (FIG. 2A), when the sample fluid is contained within acapillary. In one embodiment, the cell is made of glass, but othersubstances transparent to light in the range of about 200 to about 1,000nm or higher, such as quartz, fused silica, and organic materials suchas Teflon, nylon, plastics, such as polyvinylchloride, polystyrene, andpolyethylene, or any combination thereof, may be used without departingfrom the scope of the present invention. Although other cross-sectionalshapes (e.g., rectangular, cylindrical) may be used without departingfrom the scope of the present invention, in one embodiment the capillaryflow cell 313 has a square cross section. In another embodiment, theinterrogation space may be defined at least in part by a channel (notshown) etched into a chip (not shown). Similar considerations apply toembodiments in which two interrogation spaces are used (370 and 371 inFIG. 2B).

The interrogation space is bathed in a fluid. In one embodiment, thefluid is aqueous. In other embodiments, the fluid is non-aqueous or acombination of aqueous and non-aqueous fluids. In addition the fluid maycontain agents to adjust pH, ionic composition, or sieving agents, suchas soluble macroparticles or polymers or gels. It is contemplated thatvalves or other devices may be present between the interrogation spacesto temporarily disrupt the fluid connection. Interrogation spacestemporarily disrupted are considered to be connected by fluid.

In another embodiment of the invention, an interrogation space is thesingle interrogation space present within the flow cell 313 which isconstrained by the size of a laminar flow of the sample material withina diluent volume, also called sheath flow. In these and otherembodiments, the interrogation space can be defined by sheath flow aloneor in combination with the dimensions of the illumination source or thefield of view of the detector. Sheath flow can be configured in numerousways, including: The sample material is the interior material in aconcentric laminar flow, with the diluent volume in the exterior; thediluent volume is on one side of the sample volume; the diluent volumeis on two sides of the sample material; the diluent volume is onmultiple sides of the sample material, but not enclosing the samplematerial completely; the diluent volume completely surrounds the samplematerial; the diluent volume completely surrounds the sample materialconcentrically; the sample material is the interior material in adiscontinuous series of drops and the diluent volume completelysurrounds each drop of sample material.

In some embodiments, single molecule detectors of the invention compriseno more than one interrogation space. In some embodiments, multipleinterrogation spaces are used. Multiple interrogation spaces have beenpreviously disclosed and are incorporated by reference from U.S. patentapplication Ser. No. 11/048,660. One skilled in the art will recognizethat in some cases the analyzer will contain a plurality of distinctinterrogation spaces. In some embodiments, the analyzer contains 2, 3,4, 5, 6 or more distinct interrogation spaces.

3. Motive Force

In one embodiment of the analyzer system, the particles are movedthrough the interrogation space by a motive force. In some embodiments,the motive force for moving particles is pressure. In some embodiments,the pressure is supplied by a pump, and air pressure source, a vacuumsource, a centrifuge, or a combination thereof. In some embodiments, themotive force for moving particles is an electrokinetic force. The use ofan electrokinetic force as a motive force has been previously disclosedin a prior application and is incorporated by reference from U.S. patentapplication Ser. No. 11/048,660.

In one embodiment, pressure can be used as a motive force to moveparticles through the interrogation space of the capillary flow cell. Ina further embodiment, pressure is supplied to move the sample by meansof a pump. Suitable pumps are known in the art. In one embodiment, pumpsmanufactured for HPLC applications, such as those made by Scivax, Inc.can be used as a motive force. In other embodiments, pumps manufacturedfor microfluidics applications can be used when smaller volumes ofsample are being pumped. Such pumps are described in U.S. Pat. Nos.5,094,594, 5,730,187, 6,033,628, and 6,533,553, which discloses deviceswhich can pump fluid volumes in the nanoliter or picoliter range.Preferably all materials within the pump that come into contact withsample are made of highly inert materials, e.g., polyetheretherketone(PEEK), fused silica, or sapphire.

A motive force is necessary to move the sample through the capillaryflow cell to push the sample through the interrogation space foranalysis. A motive force is also required to push a flushing samplethrough the capillary flow cell after the sample has been passedthrough. A motive force is also required to push the sample back outinto a sample recovery vessel, when sample recovery is employed.Standard pumps come in a variety of sizes, and the proper size may bechosen to suit the anticipated sample size and flow requirements. Insome embodiments, separate pumps are used for sample analysis and forflushing of the system. The analysis pump may have a capacity ofapproximately 0.000001 mL to approximately 10 mL, or approximately 0.001mL to approximately 1 mL, or approximately 0.01 mL to approximately 0.2mL, or approximately 0.005, 0.01, 0.05, 0.1, or 0.5 mL. Flush pumps maybe of larger capacity than analysis pumps. Flush pumps may have a volumeof about 0.01 mL to about 20 mL, or about 0.1 mL to about 10 mL, orabout 0.1 mL to about 2 mL, or about or about 0.05, 0.1, 0.5, 1, 5, or10 mL. These pump sizes are illustrative only, and those of skill in theart will appreciate that the pump size may be chosen according to theapplication, sample size, viscosity of fluid to be pumped, tubingdimensions, rate of flow, temperature, and other factors well known inthe art. In some embodiments, pumps of the system are driven by steppermotors, which are easy to control very accurately with a microprocessor.

In preferred embodiments, the flush and analysis pumps are used inseries, with special check valves to control the direction of flow. Theplumbing is designed so that when the analysis pump draws up the maximumsample, the sample does not reach the pump itself. This is accomplishedby choosing the ID and length of the tubing between the analysis pumpand the analysis capillary such that the tubing volume is greater thanthe stroke volume of the analysis pump.

4. Detectors

In one embodiment, light (e.g., light in the ultra-violet, visible orinfrared range) emitted by a fluorescent label after exposure toelectromagnetic radiation is detected. The detector 309 (FIG. 1A), ordetectors (364, 365, FIG. 1B), is capable of capturing the amplitude andduration of photon bursts from a fluorescent moiety, and furtherconverting the amplitude and duration of the photon burst to electricalsignals. Detection devices such as CCD cameras, video input modulecameras, and Streak cameras can be used to produce images withcontiguous signals. In another embodiment, devices such as a bolometer,a photodiode, a photodiode array, avalanche photodiodes, andphotomultipliers which produce sequential signals may be used. Anycombination of the aforementioned detectors may also be used. In oneembodiment, avalanche photodiodes are used for detecting photons.

Using specific optics between an interrogation space 314 (FIG. 2A) andits corresponding detector 309 (FIG. 1A), several distinctcharacteristics of the emitted electromagnetic radiation can be detectedincluding: emission wavelength, emission intensity, burst size, burstduration, and fluorescence polarization. In some embodiments, thedetector 309 is a photodiode that is used in reverse bias. A photodiodeset in reverse bias usually has an extremely high resistance. Thisresistance is reduced when light of an appropriate frequency shines onthe P/N junction. Hence, a reverse biased diode can be used as adetector by monitoring the current running through it. Circuits based onthis effect are more sensitive to light than ones based on zero bias.

In one embodiment of the analyzer system, the photodiode can be anavalanche photodiode, which can be operated with much higher reversebias than conventional photodiodes, thus allowing each photo-generatedcarrier to be multiplied by avalanche breakdown, resulting in internalgain within the photodiode, which increases the effective responsiveness(sensitivity) of the device. The choice of photodiode is determined bythe energy or emission wavelength emitted by the fluorescently labeledparticle. In some embodiments, the photodiode is a silicon photodiodethat detects energy in the range of 190-1100 nm; in another embodimentthe photodiode is a germanium photodiode that detects energy in therange of 800-1700 nm; in another embodiment the photodiode is an indiumgallium arsenide photodiode that detects energy in the range of 800-2600nm; and in yet other embodiments, the photodiode is a lead sulfidephotodiode that detects energy in the range of between less than 1000 nmto 3500 nm. In some embodiments, the avalanche photodiode is asingle-photon detector designed to detect energy in the 400 nm to 1100nm wavelength range. Single photon detectors are commercially available(e.g., Perkin Elmer, Wellesley, Mass.).

In some embodiments, the detector is an avalanche photodiode detectorthat detects energy between 300 nm and 1700 nm. In one embodiment,silicon avalanche photodiodes can be used to detect wavelengths between300 nm and 1100 nm. Indium gallium arsenic photodiodes can be used todetect wavelengths between 900 nm and 1700 nm. In some embodiments, ananalyzer system can comprise at least one detector; in otherembodiments, the analyzer system can comprise at least two detectors,and each detector can be chosen and configured to detect light energy ata specific wavelength range. For example, two separate detectors can beused to detect particles that have been tagged with different labels,which upon excitation with an EM source, will emit photons with energyin different spectra. In one embodiment, an analyzer system can comprisea first detector that can detect fluorescent energy in the range of450-700 nm such as that emitted by a green dye (e.g., Alexa Fluor 546);and a second detector that can detect fluorescent energy in the range of620-780 nm such as that emitted by a far-red dye (e.g., Alexa Fluor647). Detectors for detecting fluorescent energy in the range of 400-600nm such as that emitted by blue dyes (e.g., Hoechst 33342), and fordetecting energy in the range of 560-700 nm such as that emitted by reddyes (Alexa Fluor 546 and Cy3) can also be used.

A system comprising two or more detectors can be used to detectindividual particles that are each tagged with two or more labels thatemit light in different spectra. For example, two different detectorscan detect an antibody that has been tagged with two different dyelabels. Alternatively, an analyzer system comprising two detectors canbe used to detect particles of different types, each type being taggedwith different dye molecules, or with a mixture of two or more dyemolecules. For example, two different detectors can be used to detecttwo different types of antibodies that recognize two different proteins,each type being tagged with a different dye label or with a mixture oftwo or more dye label molecules. By varying the proportion of the two ormore dye label molecules, two or more different particle types can beindividually detected using two detectors. It is understood that threeor more detectors can be used without departing from the scope of theinvention.

It should be understood by one skilled in the art that one or moredetectors can be configured at each interrogation space, whether one ormore interrogation spaces are defined within a flow cell, and that eachdetector may be configured to detect any of the characteristics of theemitted electromagnetic radiation listed above. The use of multipledetectors, e.g., for multiple interrogation spaces, has been previouslydisclosed in a prior application and is incorporated by reference herefrom U.S. patent application Ser. No. 11/048,660. Once a particle islabeled to render it detectable (or if the particle possesses anintrinsic characteristic rendering it detectable), any suitabledetection mechanism known in the art may be used without departing fromthe scope of the present invention, for example a CCD camera, a videoinput module camera, a Streak camera, a bolometer, a photodiode, aphotodiode array, avalanche photodiodes, and photomultipliers producingsequential signals, and combinations thereof. Different characteristicsof the electromagnetic radiation may be detected including: emissionwavelength, emission intensity, burst size, burst duration, fluorescencepolarization, and any combination thereof.

C. Sampling System

In a further embodiment, the analyzer system may include a samplingsystem to prepare the sample for introduction into the analyzer system.The sampling system included is capable of automatically sampling aplurality of samples and providing a fluid communication between asample container and a first interrogation space.

In some embodiments, the analyzer system of the invention includes asampling system for introducing an aliquot of a sample into the singleparticle analyzer for analysis. Any mechanism that can introduce asample may be used. Samples can be drawn up using either a vacuumsuction created by a pump or by pressure applied to the sample thatwould push liquid into the tube, or by any other mechanism that servesto introduce the sample into the sampling tube. Generally, but notnecessarily, the sampling system introduces a sample of known samplevolume into the single particle analyzer; in some embodiments where thepresence or absence of a particle or particles is detected, preciseknowledge of the sample size is not critical. In preferred embodimentsthe sampling system provides automated sampling for a single sample or aplurality of samples. In embodiments where a sample of known volume isintroduced into the system, the sampling system provides a sample foranalysis of more than about 0.0001, 0.001, 0.01, 0.1, 1, 2, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 1500, or 2000 μl.In some embodiments the sampling system provides a sample for analysisof less than about 2000, 1000, 500, 200, 100, 90, 80, 70, 60, 50, 40,30, 20, 10, 5, 2, 1, 0.1, 0.01, or 0.001 μl. In some embodiments thesampling system provides a sample for analysis of between about 0.01 and1500 μl, or about 0.1 and 1000 μl, or about 1 and 500 μl, or about 1 and100 μl, or about 1 and 50 μl, or about 1 and 201. In some embodiments,the sampling system provides a sample for analysis between about 5 μland 200 μl, or about 5 μl and about 100 μl, or about 5 μl and 501. Insome embodiments, the sampling system provides a sample for analysisbetween about 10 μl and 200 μl, or between about 10 μl and 100 μl, orbetween about 10 μl and 50 μl. In some embodiments, the sampling systemprovides a sample for analysis between about 0.5 μl and about 50 μl.

Because of the sensitivity of the methods of the present invention, verysmall sample volumes can be used. For example, the methods here can beused to measure VEGF in small sample volumes, e.g., 10 μl or less,compared to the standard sample volume of 100 μl. The present inventionenables a greater number of samples to provide quantifiable results insmall volume samples compared to other methods. For example, a lysateprepared from a typical 1 mm needle biopsy may have a volume less thanor equal to 10 μl. Using the present invention, such sample can beassayed. In some embodiments, the present invention allows the use ofsample volume under 100 μl. In some embodiments, the present inventionallows the use of sample volume under 90 μl. In some embodiments, thepresent invention allows the use of sample volume under 80 μl. In someembodiments, the present invention allows the use of sample volume under70 μl. In some embodiments, the present invention allows the use ofsample volume under 60 μl. In some embodiments, the present inventionallows the use of sample volume under 50 μl. In some embodiments, thepresent invention allows the use of sample volume under 40 μl. In someembodiments, the present invention allows the use of sample volume under30 μl. In some embodiments, the present invention allows the use ofsample volume under 25 μl. In some embodiments, the present inventionallows the use of sample volume under 20 μl. In some embodiments, thepresent invention allows the use of sample volume under 15 μl. In someembodiments, the present invention allows the use of sample volume under10 μl. In some embodiments, the present invention allows the use ofsample volume under 5 μl. In some embodiments, the present inventionallows the use of sample volume under 1 μl. In some embodiments, thepresent invention allows the use of sample volume under 0.05 μl. In someembodiments, the present invention allows the use of sample volume under0.01 μl. In some embodiments, the present invention allows the use ofsample volume under 0.005 μl. In some embodiments, the present inventionallows the use of sample volume under 0.001 μl. In some embodiments, thepresent invention allows the use of sample volume under 0.0005 μl. Insome embodiments, the present invention allows the use of sample volumeunder 0.0001 μl.

In some embodiments, the sampling system provides a sample size that canbe varied from sample to sample. In these embodiments, the sample sizemay be any one of the sample sizes described herein, and may be changedwith every sample, or with sets of samples, as desired.

Sample volume accuracy, and sample to sample volume precision of thesampling system, is required for the analysis at hand. In someembodiments, the precision of the sampling volume is determined by thepumps used, typically represented by a CV of less than about 50, 40, 30,20, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01% of sample volume. Insome embodiments, the sample to sample precision of the sampling systemis represented by a CV of less than about 50, 40, 30, 20, 10, 5, 4, 3,2, 1, 0.5, 0.1, 0.05, or 0.01%. In some embodiments, the intra-assayprecision of the sampling system is represented by a CV of less thanabout 10, 5, 1, 0.5, or 0.1%. In some embodiments, the intra-assayprecision of the sampling system shows a CV of less than about 10%. Insome embodiments, the interassay precision of the sampling system isrepresented by a CV of less than about 5%. In some embodiments, theinterassay precision of the sampling system shows a CV of less thanabout 1%. In some embodiments, the interassay precision of the samplingsystem is represented by a CV of less than about 0.5%. In someembodiments, the interassay precision of the sampling system shows a CVof less than about 0.1%.

In some embodiments, the sampling system provides low sample carryover,advantageous in that an additional wash step is not required betweensamples. Thus, in some embodiments, sample carryover is less than about1, 0.5, 0.1, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, or 0.001%. In someembodiments, sample carryover is less than about 0.02%. In someembodiments, sample carryover is less than about 0.01%.

In some embodiments the sampler provides a sample loop. In theseembodiments, multiple samples are drawn into tubing sequentially andeach is separated from the others by a “plug” of buffer. The samplestypically are read one after the other with no flushing in between.Flushing is done once at the end of the loop. In embodiments where abuffer “plug” is used, the plug may be recovered ejecting the bufferplug into a separate well of a microtiter plate.

The sampling system may be adapted for use with standard assayequipment, for example, a 96-well microtiter plate, or, preferably, a384-well plate. In some embodiments the system includes a 96 well platepositioner and a mechanism to dip the sample tube into and out of thewells, e.g., a mechanism providing movement along the X, Y, and Z axes.In some embodiments, the sampling system provides multiple samplingtubes from which samples may be stored and extracted from, when testingis commenced. In some embodiments, all samples from the multiple tubesare analyzed on one detector. In other embodiments, multiple singlemolecule detectors may be connected to the sample tubes. Samples may beprepared by steps that include operations performed on sample in thewells of the plate prior to sampling by the sampling system, or samplemay be prepared within the analyzer system, or some combination of both.

D. Sample Preparation System

Sample preparation includes the steps necessary to prepare a raw samplefor analysis. These steps can involve, by way of example, one or moresteps of: separation steps such as centrifugation, filtration,distillation, chromatography; concentration, cell lysis, alteration ofpH, addition of buffer, addition of diluents, addition of reagents,heating or cooling, addition of label, binding of label, cross-linkingwith illumination, separation of unbound label, inactivation and/orremoval of interfering compounds and any other steps necessary for thesample to be prepared for analysis by the single particle analyzer. Insome embodiments, blood is treated to separate out plasma or serum.Additional labeling, removal of unbound label, and/or dilution steps mayalso be performed on the serum or plasma sample.

In some embodiments, the analyzer system includes a sample preparationsystem that performs some or all of the processes needed to provide asample ready for analysis by the single particle analyzer. This systemmay perform any or all of the steps listed above for sample preparation.In some embodiments samples are partially processed by the samplepreparation system of the analyzer system. Thus, in some embodiments, asample may be partially processed outside the analyzer system first. Forexample, the sample may be centrifuged first. The sample may then bepartially processed inside the analyzer by a sample preparation system.Processing inside the analyzer includes labeling the sample, mixing thesample with a buffer and other processing steps that will be known toone in the art. In some embodiments, a blood sample is processed outsidethe analyzer system to provide a serum or plasma sample, which isintroduced into the analyzer system and further processed by a samplepreparation system to label the particle or particles of interest and,optionally, to remove unbound label. In other embodiments preparation ofthe sample can include immunodepletion of the sample to remove particlesthat are not of interest or to remove particles that can interfere withsample analysis. In yet other embodiments, the sample can be depleted ofparticles that can interfere with the analysis of the sample. Forexample, sample preparation can include the depletion of heterophilicantibodies, which are known to interfere with immunoassays that usenon-human antibodies to directly or indirectly detect a particle ofinterest. Similarly, other proteins that interfere with measurements ofthe particles of interest can be removed from the sample usingantibodies that recognize the interfering proteins.

In some embodiments, the sample can be subjected to solid phaseextraction prior to being assayed and analyzed. For example, a serumsample that is assayed for cAMP can first be subjected to solid phaseextraction using a c18 column to which it binds. Other proteins such asproteases, lipases and phosphatases are washed from the column, and thecAMP is eluted essentially free of proteins that can degrade orinterfere with measurements of cAMP. Solid phase extraction can be usedto remove the basic matrix of a sample, which can diminish thesensitivity of the assay. In yet other embodiments, the particles ofinterest present in a sample may be concentrated by drying orlyophilizing a sample and solubilizing the particles in a smaller volumethan that of the original sample.

In some embodiments the analyzer system provides a sample preparationsystem that provides complete preparation of the sample to be analyzedon the system, such as complete preparation of a blood sample, a salivasample, a urine sample, a cerebrospinal fluid sample, a lymph sample, aBAL sample, a biopsy sample, a forensic sample, a bioterrorism sample,and the like. In some embodiments the analyzer system provides a samplepreparation system that provides some or all of the sample preparation.In some embodiments, the initial sample is a blood sample that isfurther processed by the analyzer system. In some embodiments, thesample is a serum or plasma sample that is further processed by theanalyzer system. The serum or plasma sample may be further processed by,e.g., contacting with a label that binds to a particle or particles ofinterest; the sample may then be used with or without removal of unboundlabel.

In some embodiments, sample preparation is performed, either outside theanalysis system or in the sample preparation component of the analysissystem, on one or more microtiter plates, such as a 96-well plate.Reservoirs of reagents, buffers, and the like can be in intermittentfluid communication with the wells of the plate by means of tubing orother appropriate structures, as are well-known in the art. Samples maybe prepared separately in 96 well plates or tubes. Sample isolation,label binding and, if necessary, label separation steps may be done onone plate. In some embodiments, prepared particles are then releasedfrom the plate and samples are moved into tubes for sampling into thesample analysis system. In some embodiments, all steps of thepreparation of the sample are done on one plate and the analysis systemacquires sample directly from the plate. Although this embodiment isdescribed in terms of a 96-well plate, it will be appreciated that anyvessel for containing one or more samples and suitable for preparationof sample may be used. For example, standard microtiter plates of 384 or1536 wells may be used. More generally, in some embodiments, the samplepreparation system is capable of holding and preparing more than about5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 1000, 5000,or 10,000 samples. In some embodiments, multiple samples may be sampledfor analysis in multiple analyzer systems. Thus, in some embodiments, 2samples, or more than about 2, 3, 4, 5, 7, 10, 15 20, 50, or 100 samplesare sampled from the sample preparation system and run in parallel onmultiple sample analyzer systems.

Microfluidics systems may also be used for sample preparation and assample preparation systems that are part of analyzer systems, especiallyfor samples suspected of containing concentrations of particles highenough that detection requires smaller samples. Principles andtechniques of microfluidic manipulation are known in the art. See, e.g.,U.S. Pat. Nos. 4,979,824; 5,770,029; 5,755,942; 5,746,901; 5,681,751;5,658,413; 5,653,939; 5,653,859; 5,645,702; 5,605,662; 5,571,410;5,543,838; 5,480,614; 5,716,825; 5,603,351; 5,858,195; 5,863,801;5,955,028; 5,989,402; 6,041,515; 6,071,478; 6,355,420; 6,495,104;6,386,219; 6,606,609; 6,802,342; 6,749,734; 6,623,613; 6,554,744;6,361,671; 6,143,152; 6,132,580; 5,274,240; 6,689,323; 6,783,992;6,537,437; 6,599,436; 6,811,668 and published PCT Patent Application No.WO9955461(A1). Samples may be prepared in series or in parallel, for usein a single or multiple analyzer systems.

In some embodiments, the sample comprises a buffer. The buffer may bemixed with the sample outside the analyzer system, or it may be providedby the sample preparation mechanism. While any suitable buffer can beused, the preferable buffer has low fluorescence background, is inert tothe detectably labeled particle, can maintain the working pH and, inembodiments wherein the motive force is electrokinetic, has suitableionic strength for electrophoresis. The buffer concentration can be anysuitable concentration, such as in the range from about 1 to about 200mM. Any buffer system may be used as long as it provides for solubility,function, and delectability of the molecules of interest. In someembodiments, e.g., for application using pumping, the buffer is selectedfrom the group consisting of phosphate, glycine, acetate, citrate,acidulate, carbonate/bicarbonate, imidazole, triethanolamine, glycineamide, borate, MES, Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris Propane,BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA,EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES,CAPSO, AMP, CAPS, and CABS. The buffer can also be selected from thegroup consisting of Gly-Gly, bicine, tricine, 2-morpholineethanesulfonic acid (MES),4-morpholine propanesulfonic acid (MOPS) and2-amino-2-methyl-1-propanol hydrochloride (AMP). A useful buffer is 2 mMTris/borate at pH 8.1, but Tris/glycine and Tris/HCl are alsoacceptable. Other buffers are as described herein.

Buffers useful for electrophoresis are disclosed in a prior applicationand are incorporated by reference herein from U.S. patent applicationSer. No. 11/048,660.

E. Sample recovery

One highly useful feature of embodiments of the analyzers and analysissystems of the invention is that the sample can be analyzed withoutconsuming it. This can be especially important when sample materials arelimited. Recovering the sample also allows one to do other analyses orreanalyze it. The advantages of this feature for applications wheresample size is limited and/or where the ability to reanalyze the sampleis desirable, e.g., forensic, drug screening, and clinical diagnosticapplications, will be apparent to those of skill in the art.

Thus, in some embodiments, the analyzer system of the invention furtherprovides a sample recovery system for sample recovery after analysis. Inthese embodiments, the system includes mechanisms and methods by whichthe sample is drawn into the analyzer, analyzed and then returned, e.g.,by the same path, to the sample holder, e.g., the sample tube. Becauseno sample is destroyed and because it does not enter any of the valvesor other tubing, it remains uncontaminated. In addition, because all thematerials in the sample path are highly inert, e.g., PEEK, fused silica,or sapphire, there is little contamination from the sample path. The useof the stepper motor controlled pumps (particularly the analysis pump)allows precise control of the volumes drawn up and pushed back out. Thisallows complete or nearly complete recovery of the sample with little ifany dilution by the flush buffer. Thus, in some embodiments, more thanabout 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9% of the sample is recovered after analysis. In some embodiments,the recovered sample is undiluted. In some embodiments, the recoveredsample is diluted less than about 1.5-fold, 1.4-fold, 1.3-fold,1.2-fold, 1.1-fold, 1.05-fold, 1.01-fold, 1.005-fold, or 1.001-fold.

For sampling and/or sample recovery, any mechanism for transporting aliquid sample from a sample vessel to the analyzer may be used. In someembodiments the inlet end of the analysis capillary has attached a shortlength of tubing, e.g., PEEK tubing that can be dipped into a samplecontainer, e.g., a test tube or sample well, or can be held above awaste container. When flushing, to clean the previous sample from theapparatus, this tube is positioned above the waste container to catchthe flush waste. When drawing a sample in, the tube is put into thesample well or test tube. Typically the sample is drawn in quickly, andthen pushed out slowly while observing particles within the sample.Alternatively, in some embodiments, the sample is drawn in slowly duringat least part of the draw-in cycle; the sample may be analyzed whilebeing slowly drawn in. This can be followed by a quick return of thesample and a quick flush. In some embodiments, the sample may beanalyzed both on the inward (draw-in) and outward (pull out) cycle,which improves counting statistics, e.g., of small and dilute samples,as well as confirming results, and the like. If it is desired to savethe sample, it can be pushed back out into the same sample well it camefrom, or to another. If saving the sample is not desired, the tubing ispositioned over the waste container.

VI. METHODS USING HIGHLY SENSITIVE ANALYSIS OF MOLECULES

The systems, system kits, and methods of the present invention makepossible measurement of molecules in samples at concentrations far lowerthan previously measured. The high sensitivity of the instruments, kits,and methods of the invention allows the establishment of markers, e.g.,biological markers, that have not previously been possible because of alack of sensitivity of detection. The invention also includes the use ofthe compositions and methods described herein for the discovery of newmarkers.

There are numerous markers currently available which, while potentiallyof use in determining a biological state, are not currently of practicaluse because their lower ranges are unknown. In some cases, abnormallyhigh levels of the marker are detectable by current methodologies, butnormal ranges have not been established. In some cases, upper normalranges of the marker are detectable, but not lower normal ranges, orlevels below normal. In some cases, for example, markers specific totumors, or markers of infection, any level of the marker indicates thepotential presence of the biological state, and enhancing sensitivity ofdetection is an advantage for early diagnosis. In some cases, the rateof change, or lack of change, in the concentration of the marker overmultiple timepoints provides the most useful information, but presentmethods of analysis do not permit determination of levels of the markerat timepoint sampling in the early stages of a condition, when it istypically at its most treatable. In many cases, the marker may bedetected at clinically useful levels only through the use of cumbersomemethods that are not practical or useful in a clinical setting, such asmethods that require complex sample treatment and time-consuminganalysis.

In addition, there are potential markers of biological states that existin sufficiently low concentrations that their presence remains extremelydifficult or impossible to detect by current methods.

The analytical methods and compositions of the present invention providelevels of sensitivity and precision that allow the detection of markersfor biological states at concentrations at which the markers have beenpreviously undetectable, thus allowing the “repurposing” of such markersfrom confirmatory markers, or markers useful only in limited researchsettings, to diagnostic, prognostic, treatment-directing, or other typesof markers useful in clinical settings and/or in large-scale clinicalsettings such as clinical trials. Such methods allow, e.g., thedetermination of normal and abnormal ranges for such markers.

The markers thus repurposed can be used for, e.g., detection of normalstate (normal ranges), detection of responder/non-responder (e.g., to atreatment, such as administration of a drug); early disease orpathological occurrence detection (e.g., detection of cancer in itsearliest stages, early detection of cardiac ischemia); disease staging(e.g., cancer); disease monitoring (e.g., diabetes monitoring,monitoring for recurrence of cancer after treatment); study of diseasemechanism; and study of treatment toxicity, such as toxicity of drugtreatments (e.g., cardiotoxicity).

A. Methods

The invention thus provides methods and compositions for the sensitivedetection of markers, and further methods of establishing values fornormal and abnormal levels of the markers. In further embodiments, theinvention provides methods of diagnosis, prognosis, and/or treatmentselection based on values established for the markers. The inventionalso provides compositions for use in such methods, e.g., detectionreagents for the ultrasensitive detection of markers.

In some embodiments, the invention provides a method of establishing amarker for a biological state, by establishing a range of concentrationsfor the marker in biological samples obtained from a first population bymeasuring the concentrations of the marker the biological samples bydetecting single molecules of the marker, e.g., by detecting a labelthat has been attached to a single molecule of the marker. In someembodiments, the marker is a polypeptide or small molecule. The samplesmay be any sample type described herein, e.g., blood, plasma, serum, orurine.

The method may utilize samples from a first population where thepopulation is a population that does not exhibit the biological state.In the case where the biological state is a disease state, the firstpopulation may be a population that does not exhibit the disease, e.g.,a “normal” population. In some embodiments the method may furthercomprise establishing a range of range of levels for the marker inbiological samples obtained from a second population, where the membersof the second population exhibit the biological state, by measuring theconcentrations of the marker the biological samples by detecting singlemolecules of the marker. In some embodiments, e.g., cross-sectionalstudies, the first and second populations are different. In someembodiments, at least one member of the second population is a member ofthe first population, or at least one member of said the population is amember of the second population. In some embodiments, e.g., longitudinalstudies, substantially all the members of the second population aremembers of the first population who have developed the biological state,e.g., a disease or pathological state.

The detecting of single molecules of the marker is performed using amethod as described herein, e.g., a method with a limit of detection forsaid marker of less than about 1000, 100, 50, 20, 10, 5, 1, 0.5, 0.1,0.05, 0.01, 0.005, or 0.001 femtomolar of the marker in the samples, bydetecting single molecules of the marker. In some embodiments, the limitof detection of said marker is than about 100, 50, 20, 10, 5, 1, 0.5,0.1, 0.05, 0.01, 0.005, or 0.001 pg/ml of the marker in the samples, bydetecting single molecules of the marker.

The biological state may be a phenotypic state; a condition affectingthe organism; a state of development; age; health; pathology; disease;disease process; disease staging; infection; toxicity; or response tochemical, environmental, or drug factors (such as drug responsephenotyping, drug toxicity phenotyping, or drug effectivenessphenotyping).

In some embodiments, the biological state is a pathological state,including but not limited to inflammation, abnormal cell growth, andabnormal metabolic state. In some embodiments, the state is a diseasestate. Disease states include, but are not limited to, cancer,cardiovascular disease, inflammatory disease, autoimmune disease,neurological disease, infectious disease and pregnancy relateddisorders. In some embodiments the state is a disease stage state, e.g.,a cancer disease stage state.

The methods may also be used for determination of a treatment responsestate. In some embodiments, the treatment is a drug treatment. Theresponse may be a therapeutic effect or a side effect, e.g., an adverseeffect. Markers for therapeutic effects will be based on the disease orcondition treated by the drug. Markers for adverse effects typicallywill be based on the drug class and specific structure and mechanism ofaction and metabolism. A common adverse effect is drug toxicity. Anexample is cardiotoxicity, which can be monitored by the marker cardiactroponin. In some embodiments one or more markers for the disease stateand one or more markers for one or more adverse effects of a drug aremonitored, typically in a population that is receiving the drug. Samplesmay be taken at intervals and the respective values of the markers inthe samples may be evaluated over time.

The detecting of single molecules of the marker may comprise labelingthe marker with a label comprising a fluorescent moiety capable ofemitting at least about 200 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, where the laser isfocused on a spot not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the fluorescent moiety comprises a molecule that comprises at least onesubstituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance. In some embodiments, the fluorescent moiety maycomprise a dye selected from the group consisting of Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or Alexa Fluor 700. Insome embodiments, the moiety comprises Alexa Fluor 647. In someembodiments, the label further comprises a binding partner for themarker, e.g., an antibody specific for said marker, such as a polyclonalantibody or a monoclonal antibody. Binding partners for a variety ofmarkers are described herein.

The method may further include establishing a threshold level for themarker based on the first range, or the first and second ranges, wherethe presence of marker in a biological sample from an individual at alevel above or below the threshold level indicates an increasedprobability of the presence of the biological state in said individual.An example of a threshold determined for a normal population is thesuggested threshold for cardiac troponin of greater than the 99thpercentile value in a normal population. See Example 3. Other thresholdlevels may be determined empirically, i.e., based on data from the firstand second populations regarding marker levels and the presence,absence, severity, rate of progression, rate of regression, and thelike, of the biological state being monitored. It will be appreciatedthat threshold levels may be established at either end of a range, e.g.,a minimum below which the concentration of the marker in a sampleindicates an increased probability of a biological state, and/or amaximum above which the concentration of the marker in a sampleindicates an increased probability of a biological state. In someembodiments, a risk stratification may be produced in which two or moreranges of marker concentrations correspond to two or more levels ofrisk. Other methods of analyzing data from two populations and formarkers and producing clinically-relevant values for use by, e.g.,physicians and other health care professionals, are well-known in theart.

For some biological markers, the presence of any marker at all is anindication of a disease or pathological state, and the threshold isessentially zero. An example is the use of prostate specific antigen(PSA) to monitor cancer recurrence after removal of the prostate gland.As PSA is produced only by the prostate gland, and as the prostate glandand all tumors are presumed to be removed, PSA after removal is zero.Appearance of PSA at any level signals a possible recurrence of thecancer, e.g., at a metastatic site. Thus, the more sensitive the methodof detection, the earlier an intervention may be made should suchrecurrence occur.

Other evaluations of marker concentration may also be made, such as in aseries of samples, where change in value, rate of change, spikes,decrease, and the like may all provide useful information fordetermination of a biological state. In addition, panels of markers maybe used if it is found that more than one marker provides informationregarding a biological state. If panels of markers are used, the markersmay be measured separately in separate samples (e.g., aliquots of acommon sample) or simultaneously by multiplexing. Examples of panels ofmarkers and multiplexing are given in, e.g., U.S. patent applicationSer. No. 11/048,660.

The establishment of such markers and, e.g., reference ranges for normaland/or abnormal states, allow for sensitive and precise determination ofthe biological state of an organism. Thus, in some embodiments, theinvention provides a method for detecting the presence or absence of abiological state of an organism, comprising i) measuring theconcentration of a marker in a biological sample from the organism,wherein said marker is a marker established through establishing a rangeof concentrations for said marker in biological samples obtained from afirst population by measuring the concentrations of the marker thebiological samples by detecting single molecules of the marker; and ii)determining the presence of absence of said biological state based onsaid concentration of said marker in said organism.

In some embodiments, the invention provides a method for detecting thepresence or absence of a biological state in an organism, comprising i)measuring the concentrations of a marker in a plurality of biologicalsamples from said organism, wherein said marker is a marker establishedthrough establishing a range of concentrations for said marker inbiological samples obtained from a first population by measuring theconcentrations of the marker the biological samples by detecting singlemolecules of the marker; and ii) determining the presence of absence ofsaid biological state based on said concentrations of said marker insaid plurality of samples. In some embodiments, the samples are ofdifferent types, e.g., are samples from different tissue types. In thiscase, the determining is based on a comparison of the concentrations ofsaid marker in said different types of samples. More commonly, thesamples are of the same type, and the samples are taken at intervals.The samples may be any sample type described herein, e.g., blood,plasma, or serum; or urine. Intervals between samples may be minutes,hours, days, weeks, months, or years. In an acute clinical setting, theintervals may be minutes or hours. In settings involving the monitoringof an individual, the intervals may be days, weeks, months, or years.

In many cases, the biological state whose presence or absence is to bedetected is a disease phenotype. Thus, in one embodiment, a phenotypicstate of interest is a clinically diagnosed disease state. Such diseasestates include, for example, cancer, cardiovascular disease,inflammatory disease, autoimmune disease, neurological disease,respiratory disease, infectious disease and pregnancy related disorders.

Cancer phenotypes are included in some aspects of the invention.Examples of cancer herein include, but are not limited to: breastcancer, skin cancer, bone cancer, prostate cancer, liver cancer, lungcancer, brain cancer, cancer of the larynx, gallbladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, non-small cell lungcarcinoma gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms,intestinal ganglloneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicand other sarcoma, malignant hypercalcemia, renal cell tumor,polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias,lymphomas, malignant melanomas, epidermoid carcinomas, and othercarcinomas and sarcomas.

Cardiovascular disease may be included in other applications of theinvention. Examples of cardiovascular disease include, but are notlimited to, congestive heart failure, high blood pressure, arrhythmias,atherosclerosis, cholesterol, Wolff-Parkinson-White Syndrome, long QTsyndrome, angina pectoris, tachycardia, bradycardia, atrialfibrillation, ventricular fibrillation, congestive heart failure,myocardial ischemia, myocardial infarction, cardiac tamponade,myocarditis, pericarditis, arrhythmogenic right ventricular dysplasia,hypertrophic cardiomyopathy, Williams syndrome, heart valve diseases,endocarditis, bacterial, pulmonary atresia, aortic valve stenosis,Raynaud's disease, cholesterol embolism, Wallenberg syndrome,Hippel-Lindau disease, and telangiectasis.

Inflammatory disease and autoimmune disease may be included in otherembodiments of the invention. Examples of inflammatory disease andautoimmune disease include, but are not limited to, rheumatoidarthritis, non-specific arthritis, inflammatory disease of the larynx,inflammatory bowel disorder, psoriasis, hypothyroidism (e.g., Hashimotothyroidism), colitis, Type 1 diabetes, pelvic inflammatory disease,inflammatory disease of the central nervous system, temporal arteritis,polymyalgia rheumatica, ankylosing spondylitis, polyarteritis nodosa,Reiter's syndrome, scleroderma, systemic lupus and erythematosus.

The methods and compositions of the invention can also providelaboratory information about markers of infectious disease includingmarkers of Adenovirus, Bordella pertussis, Chlamydia pneumoiea,Chlamydia trachomais, Cholera Toxin, Cholera Toxin β, Campylobacterjejuni, Cytomegalovirus, Diptheria Toxin, Epstein-Barr NA, Epstein-BarrEA, Epstein-Barr VCA, Helicobacter Pylori, Hepatitis B virus (HBV) Core,Hepatitis B virus (HBV) Envelope, Hepatitis B virus (HBV) Survace (Ay),Hepatitis C virus (HCV) Core, Hepatitis C virus (HCV) NS3, Hepatitis Cvirus (HCV) NS4, Hepatitis C virus (HCV) NS5, Hepititis A, Hepititis D,Hepatitis E virus (HEV) orf2 3 KD, Hepatitis E virus (HEV) orf2 6 KD,Hepatitis E virus (HEV) orf3 3 KD, Human immunodeficiency virus (HIV)-1p24, Human immunodeficiency virus (HIV)-1 gp41, Human immunodeficiencyvirus (HIV)-1 gp120, Human papilloma virus (HPV), Herpes simplex virusHSV-1/2, Herpes simplex virus HSV-1 gD, Herpes simplex virus HSV-2 gG,Human T-cell leukemia virus (HTLV)-1/2, Influenza A, Influenza A H3N2,Influenza B, Leishmanina donovani, Lyme disease, Mumps, M. pneumoniae,M. teberculosis, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3,Polio Virus, Respiratory syncytial virus (RSV), Rubella, Rubeola,Streptolysin O, Tetanus Toxin, T. pallidum 15 kd, T. pallidum p47, T.cruzi, Toxoplasma, and Varicella Zoater.

Detection and monitoring of cancers often depends on the use of crudemeasurements of tumor growth, such as visualization of the tumor itself,that are either inaccurate or that must reach high levels before theybecome detectable, e.g., in a practical clinical setting by presentmethods. At the point of detection, the tumor has often grown tosufficient size that intervention is unlikely to occur beforemetastasis. For example, detection of lung cancer by X-ray requires atumor of >1 cm in diameter, and by CT scan of >2-3 mm. Alternatively, abiomarker of tumor growth may be used, but, again, often the tumor iswell-advanced by the time the biomarker is detectable at levelsaccessible to current clinical technology. Furthermore, afterintervention (e.g., surgery, chemotherapy, or radiation to shrink orremove the tumor or tumors), it is often not possible to measure thetumor marker with sufficient sensitivity to determine if there has beena recurrence of the cancer until residual disease has progressed to thepoint where further intervention is unlikely to be successful. Using theanalyzers, systems, and methods of the present invention, it is possibleto both detect onset of tumor growth and return of tumor growth at apoint where intervention is more likely to be successful, e.g., due tolower probability of metastasis. Markers for cancer that can be detectedat levels not previously shown include markers disclosed herein.Examples of assays for the detection of markers that can be repurposedto diagnostic markers include TGFβ, Akt1, Fas ligand and IL-6, asdescribed herein.

B. Exemplary markers

The instruments, labels, and methods of the invention have been used toestablish ranges for markers in, e.g., serum and urine, at levels 10- to100-fold lower than previous levels, or lower. The markers areindicative of a wide variety of biological states, e.g., cardiac diseaseand cardiotoxicity (troponin), infection (TREM-1), inflammation andother conditions (LTE4, IL-6 and IL-8), asthma (LTE4), cancer (Akt1,TGF-beta, Fas ligand), and allograft rejection and degenerative disease(Fas ligand).

Markers include protein and non-protein markers. The markers aredescribed briefly here and procedures and results given in the Examples.

1. Cardiac damage

Cardiac troponin is an example of a marker that is previously detectableonly in abnormally high amounts. Cardiac troponin is a marker of cardiacdamage, useful in diagnosis, prognosis, and determination of method oftreatment in a number of diseases and conditions, e.g., acute myocardialinfarct (AMI). In addition, cardiac troponin is a useful marker ofcardiotoxicity due to treatment, e.g., drug treatment.

The troponin complex in muscle consists of troponin I, C and T. TroponinC exists as two isoforms, one from cardiac and slow-twitch muscle andone from fast-twitch muscle; because it is found in virtually allstriated muscle, its use as a specific marker is limited. In contrast,troponin I and T are expressed as different isoforms in slow-twitch,fast-twitch and cardiac muscle. The unique cardiac isoforms of troponinI and T allow them to be distinguished immunologically from the othertroponins of skeletal muscle. Therefore, the release into the blood ofcardiac troponin I and T is indicative of damage to cardiac muscle, andprovides the basis for their use as diagnostic or prognostic markers, orto aid in determination of treatment.

Currently used markers for cardiac damage suffer disadvantages thatlimit their clinical usefulness. Cardiac enzyme assays have formed thebasis for determining whether or not there is damage to the cardiacmuscle. Unfortunately, the standard creatine kinase-MB (CK-MB) assay isnot reliable in excluding infarction until 10 to 12 hours after theonset of chest pain. Earlier diagnosis would have very specificadvantages with regard to fibrinolytic therapy and triage.

Because the level of troponin found in the circulation of healthyindividuals is very low, and cardiac specific troponins do not arisefrom extra-cardiac sources, the troponins are very sensitive andspecific markers of cardiac injury. In addition to cardiac infarct, anumber of other conditions can cause damage to the heart muscle, andearly detection of such damage would prove useful to clinicians.However, present methods of detection and quantitation of cardiactroponin do not possess sufficient sensitivity to detect the release ofcardiac troponin into the blood until levels have reached abnormallyhigh concentrations, e.g., 0.1 ng/ml or greater.

The methods and compositions of the invention thus include methods andcompositions for the highly sensitive detection and quantitation ofcardiac troponin, and compositions and methods for diagnosis, prognosis,and/or determination of treatment based on such highly sensitivedetection and quantitation. A standard curve for cardiac troponin I wasestablished with a limit of detection less than about 1 pg/ml (Example1). Levels of cardiac troponin I were established in normal individualsand a threshold value at the 99^(th) percentile of normal established(Example 3). Serial samples from individuals who suffered acutemyocardial infarct were analyzed, and time courses for cardiac troponinI concentrations, including deviations from baseline, were determined(Example 4). Thus, cardiac troponin I serves as an example of a markerthat can be detected by the systems and methods of the invention atlevels to provide diagnostic and prognostic information of use inclinical and research settings. See also U.S. patent application Ser.No. 11/784,213, filed Mar. 5, 2008 and entitled “Highly Sensitive Systemand Methods for Analysis of Troponin,” which is incorporated byreference herein in its entirety.

Cardiac troponin-I (cTnI) is specific to cardiomyocytes and is releasedinto blood following heart damage. Extensive studies have shown thatcTnI is slowly released from damaged cardiomyocytes and often requires4-8 hours post-trauma to be detectable. Measurement of cTnIconcentrations in plasma/serum are the standard of care for diagnosingnon-STEMI acute myocardial infarction (AMI). In addition this biomarkerhas been widely accepted in pre-clinical and clinical drug developmentsettings as an indicator of myocardial damage and hence heart damage.TnI is accepted as a biomarker to assess potential cardiotoxicity ofexperimental therapies. It is extensively studied in pre-clinicalsetting and included in clinical drug development programs whenpreclinical data suggests a potential of cardiac-related adverse events.

Even though cTnI is used as the standard of care for diagnosing AMI, aswell as in pre-clinical and clinical development, until recently itsconcentration in the plasma of apparently healthy humans and preclinicalanimal models had not been reported. Thus it was impossible to benchmarka “normal” level within a given animal or human and measure smallincreases (velocity) of cTnI which might be associated with subtlecardiac damage. Furthermore, many assays do not equally quantify cTnIacross different species and require large plasma sample volumes,limiting their use in pre-clinical settings, especially in rodent modelsystems. Using the methods of the present invention, normal levels ofendogenous cTnI and small changes in plasma cTnI can be quantified inhumans, rats, dogs and monkeys providing previously intractable answersaround cardiomyocyte pathophysiology. See Examples 1-4.

In some embodiments, the cTnI assay of the present invention is used to:(1) define the concentration of plasma and serum cTnI in healthy humans,rats, dogs and monkeys; (2) identify AMIs earlier; (3) measure heartdamage earlier under physical stress or known cardiotoxins; and/or (4)study cTnI concentrations in a single rat using only 10 μL plasma. Inother embodiments, the cTnI assay of the present invention is used to:(1) measure the potential cardiac safety and dosing of therapeutics inboth pre-clinical and clinical settings; (2) perform studies usingindividual small animals or precious samples, when sample volume is anissue; (3) design more robust clinical and preclinical studies whenvelocity of cTnI concentration change from a baseline normal level isused as an endpoint; (4) understand how cTnI levels change from normallevels in a variety of cardiac-related diseases; and/or (5) understandthe utility of cTnI as a biomarker to serve as a surrogate endpoint forclinical events.

In some embodiments, the method is capable of detecting cTnI at a limitof detection of less than about 100, 80, 60, 50, 30, 20, 10, 5, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less thanabout 100 pg/ml. In some embodiments, the method is capable of detectingthe cTnI at a limit of detection of less than about 100 pg/ml. In someembodiments, the method is capable of detecting the cTnI a limit ofdetection of less than about 80 pg/ml. In some embodiments, the methodis capable of detecting the cTnI a limit of detection of less than about60 pg/ml. In some embodiments, the method is capable of detecting thecTnI a limit of detection of less than about 50 pg/ml. In someembodiments, the method is capable of detecting the cTnI a limit ofdetection of less than about 30 pg/ml. In some embodiments, the methodis capable of detecting the cTnI a limit of detection of less than about25 pg/ml. In some embodiments, the method is capable of detecting thecTnI a limit of detection of less than about 10 pg/ml. In someembodiments, the method is capable of detecting the cTnI a limit ofdetection of less than about 5 pg/ml. In some embodiments, the method iscapable of detecting the cTnI a limit of detection of less than about 1pg/ml. In some embodiments, the method is capable of detecting the cTnIa limit of detection of less than about 0.5 pg/ml. In some embodiments,the method is capable of detecting the cTnI at a limit of detection ofless than about 0.1 pg/ml. In some embodiments, the method is capable ofdetecting the cTnI at a limit of detection of less than about 0.05pg/ml. In some embodiments, the method is capable of detecting the cTnIat a limit of detection of less than about 0.01 pg/ml. In someembodiments, the method is capable of detecting the cTnI at a limit ofdetection of less than about 0.005 pg/ml. In some embodiments, themethod is capable of detecting the cTnI at a limit of detection of lessthan about 0.001 pg/ml. In some embodiments, the method is capable ofdetecting the cTnI at a limit of detection of less than about 0.0005pg/ml. In some embodiments, the method is capable of detecting the cTnIat a limit of detection of less than about 0.0001 pg/ml.

2. Infection

Recent reports have established TREM-1 as a biomarker of bacterial orfungal infections. See, e.g., Bouchon et al. (2000) J. Immunol.164:4991-95; Colonna (2003) Nat. Rev. Immunol. 3:445-53; Gibot et al.(2004) N. Engl. J. Med. 350:451-58; Gibot et al. (2004) Ann. Intern.Med. 141:9-15. Assays of the invention suggest that TREM-1 may routinelybe measured at a concentration of 100 fM or less. See Example 9.

In some embodiments, the method is capable of detecting TREM-1 at alimit of detection of less than about 100, 80, 60, 50, 30, 20, 10, 5, 1,0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., lessthan about 100 pg/ml. In some embodiments, the method is capable ofdetecting the TREM-1 at a limit of detection of less than about 100pg/ml. In some embodiments, the method is capable of detecting theTREM-1 a limit of detection of less than about 80 pg/ml. In someembodiments, the method is capable of detecting the TREM-1 a limit ofdetection of less than about 60 pg/ml. In some embodiments, the methodis capable of detecting the TREM-1 a limit of detection of less thanabout 50 pg/ml. In some embodiments, the method is capable of detectingthe TREM-1 a limit of detection of less than about 30 pg/ml. In someembodiments, the method is capable of detecting the TREM-1 a limit ofdetection of less than about 25 pg/ml. In some embodiments, the methodis capable of detecting the TREM-1 a limit of detection of less thanabout 10 pg/ml. In some embodiments, the method is capable of detectingthe TREM-1 a limit of detection of less than about 5 pg/ml. In someembodiments, the method is capable of detecting the TREM-1 a limit ofdetection of less than about 1 pg/ml. In some embodiments, the method iscapable of detecting the TREM-1 a limit of detection of less than about0.5 pg/ml. In some embodiments, the method is capable of detecting theTREM-1 at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the TREM-1 at a limit ofdetection of less than about 0.05 pg/ml. In some embodiments, the methodis capable of detecting the TREM-1 at a limit of detection of less thanabout 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the TREM-1 at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting theTREM-1 at a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the TREM-1 at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the TREM-1 at a limit of detection ofless than about 0.0001 pg/ml.

3. Cytokines

The normal level of many cytokines, chemokines and growth factors is notknown primarily because of the inability of existing technology todetect levels that are below those found in samples from diseasedpatients. For example, the basal level of other cytokines such as IL-10,TNF-alpha, IL-4, IL-1beta, IL-2, IL-12 and IFN-gamma cannot be detectedby routine assays that are performed in a clinical setting, whereas theanalyzer systems of the invention can readily determine the level ofthese and other cytokines. Knowing the level of cytokines and growthfactors aids clinicians with the diagnosis, prognosis and treatment of avariety of diseases including cancer, and respiratory, infectious, andcardiovascular diseases. Early cytokine detection to monitor normal anddisease states in clinical specimens can be achieved using the analyzersystems of the invention to analyze samples such as plasma, serum, andurine as well as other fluid samples to provide for better translationalmedicine. For example, determining levels of cytokines for which anormal range of concentration is not known, would aid clinicians withdiagnosis and treatment of the following conditions and diseases. BoneMorphogenetic Proteins would be useful to monitor the treatment forfractures, spinal fusions, orthopedic surgery, and oral surgery;Interleukin-10 (IL-10) would be useful for detecting and monitoring forthe presence of cancers including non-Hodgkin's lymphoma, multiplemyeloma, melanoma, and ovarian cancer, as well as for detecting andmonitoring the effect of anti-inflammatory therapy, organtransplantation, immunodeficiencies, and parasitic infections;Interleukin-11 (IL-11) is useful for the detection and monitoring forthe presence of cancers such as breast cancer; Interleukin-12 (IL-12)for cancer and HIV infections; TNFα, an inflammatory cytokine, alone orin combination with IL-6, can be used as a good predictor of sepsis,acute pancreatitis, tuberculosis, and autoimmune disease such asrheumatoid arthritis and lupus.

Alternatively, databases may already exist for normal and abnormalvalues but present methods may not be practical for screeningindividuals on a routine basis to determine with sufficient sensitivitywhether the value of the individual for the marker is within the normalrange. For example, most present methods for the determination of IL-6concentration in a sample are capable of detecting IL-6 only down to aconcentration of about 5 pg/ml; the normal range of IL-6 values is about1 to about 10 pg/ml; hence, present methods are able to detect IL-6 onlyin the upper part of normal ranges. In contrast, the analyzers andanalyzer systems of the invention allow the detection of IL-6 down to aconcentration below about 0.01 pg/ml, or less than one-tenth toone-hundreth of normal range values. Thus, the analyzers and analyzersystems of the invention allow a far broader and more nuanced databaseto be produced for a biomarker, e.g., for IL-6, and also allow screeningfor that biomarker both within and outside of the normal range, allowingearlier detection. Thus, the analyzers and analyzer systems of theinvention allow a far broader and more nuanced database to be producedfor a biomarker, e.g., for IL-6, and also allow screening for thatbiomarker both within and outside of the normal range, allowing earlierdetection of conditions in which the biomarker, e.g., IL-6, isimplicated.

a. Interleukin 1

IL-1α and -β are pro-inflammatory cytokines involved in immune defenseagainst infection, and are part of the IL-1 superfamily of cytokines.Both IL-1α and IL-1β are produced by macrophages, monocytes anddendritic cells. IL-1 is involved in various immune responses with aprimary role in inflammation, making IL-1 a target for RheumatoidArthritis (RA). IL-1α and IL-1β are produced as precursor peptides,which are proteolytically processed and released in response to cellinjury, and thus induce apoptosis. IL-1β production in peripheral tissuehas also been associated with hyperalgesia (increased sensitivity topain) associated with fever.

Amgen currently markets Kineret (anakinra), a synthetic form of thehuman interleukin-1 receptor antagonist (IL-1Ra). IL-1Ra blocks thebiologic activity of IL-1 alpha and beta by competitively inhibitingIL-1 from binding to the interleukin-1 type I receptor (IL1-RI), whichis expressed in a wide variety of tissues and organs. IL-1Ra inhibitsthe biological activities of IL-1 both in vitro and in vivo, and hasbeen shown to be effective in animal models of septic shock, rheumatoidarthritis, graft versus host disease, stroke, and cardiac ischemia. Alsoin the Amgen pipeline is AMG 108, a fully human monoclonal antibody thattargets inhibition of the action of interleukin-1 (IL-1). A Phase 2clinical study is under way to assess long-term safety of treatingrheumatoid arthritis with AMG 108.

i. Interleukin 1, Alpha (IL-1α)

The broad involvement of inflammation in human disease ensures that thisprotein will remain an attractive diagnostic target. Elevated levels ofIL-1α will continue to be a diagnostic target for inflammatory diseaseslike rheumatoid arthritis. Thus, there is a need to develop assays withsensitivity to quantify low normal levels of IL-1α in order todifferentiate between low and high levels of IL-1α which indicatedisease. Also, there is a need to evaluate the potential of IL-1α as atherapeutic drug target to decrease elevated levels of IL-1α as atreatment for IL-1α associated disease. This will present a need todetect velocity of decreasing IL-1α levels to evaluate effectiveness anddosing of therapies. This may prevent adverse events like Neutropeniathat develop after co-administration of drugs targeted to inflammatorycytokine pathways, like Kineret (IL-1Ra antagonist) and enteracept(TNF-alpha antagonist). To meet these goals, it is essential to have anassay that can detect IL-1α to below normal levels in human plasma.

The present invention provides an IL-1α assay sensitive enough toquantify IL-1α concentration in plasma from healthy, normal humansubjects with previously unattainable levels of accuracy and precision.See Example 23. It enables differentiation between IL-1α concentrationsin healthy and diseased states, allowing efficient pre-clinical andclinical study design. The IL-1α assay increases the utility of IL-1α byallowing quantification at very low levels and differentiation betweensmall changes in concentration that can provide insights into drugefficacy or disease progression. The IL-1α assay enables the accuratequantification of IL-1α in human plasma with a broad dynamic range. Invarious embodiments, the assay allows investigators to: (1) measure theefficacy and dosing of therapeutics designed to interfere with the IL-1mediated inflammatory response, such as Kineret; (2) design more robustclinical and preclinical studies when IL-1α concentration can be used asa therapeutic endpoint, as in the clinical trial of AMG 108; and/or (3)understand how IL-1α levels change in patients as they transition from ahealthy to a diseased state.

In some embodiments, the method is capable of detecting IL-1α at a limitof detection of less than about 100, 80, 60, 50, 30, 20, 10, 5, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less thanabout 100 pg/ml. In some embodiments, the method is capable of detectingthe IL-1α at a limit of detection of less than about 100 pg/ml. In someembodiments, the method is capable of detecting the IL-1α a limit ofdetection of less than about 80 pg/ml. In some embodiments, the methodis capable of detecting the IL-1α a limit of detection of less thanabout 60 pg/ml. In some embodiments, the method is capable of detectingthe IL-1α a limit of detection of less than about 50 pg/ml. In someembodiments, the method is capable of detecting the IL-1α a limit ofdetection of less than about 30 pg/ml. In some embodiments, the methodis capable of detecting the IL-1α a limit of detection of less thanabout 25 pg/ml. In some embodiments, the method is capable of detectingthe IL-1α a limit of detection of less than about 10 pg/ml. In someembodiments, the method is capable of detecting the IL-1α a limit ofdetection of less than about 5 pg/ml. In some embodiments, the method iscapable of detecting the IL-1α a limit of detection of less than about 1pg/ml. In some embodiments, the method is capable of detecting the IL-1αa limit of detection of less than about 0.5 pg/ml. In some embodiments,the method is capable of detecting the IL-1α at a limit of detection ofless than about 0.1 pg/ml. In some embodiments, the method is capable ofdetecting the IL-1α at a limit of detection of less than about 0.05pg/ml. In some embodiments, the method is capable of detecting the IL-1αat a limit of detection of less than about 0.01 pg/ml. In someembodiments, the method is capable of detecting the IL-1α at a limit ofdetection of less than about 0.005 pg/ml. In some embodiments, themethod is capable of detecting the IL-1α at a limit of detection of lessthan about 0.001 pg/ml. In some embodiments, the method is capable ofdetecting the IL-1α at a limit of detection of less than about 0.0005pg/ml. In some embodiments, the method is capable of detecting the IL-1αat a limit of detection of less than about 0.0001 pg/ml.

ii. Interleukin 1, Beta (IL-1β)

Like IL-1α, the broad involvement of inflammation in human diseaseensures that IL-1β will remain an attractive diagnostic target. Elevatedlevels of IL-1β will continue to be a diagnostic target for inflammatorydiseases like rheumatoid arthritis. Thus, there is a need to developassays with sensitivity to quantify low normal levels of IL-1β in orderto differentiate between low and high levels of IL-1β which indicatedisease. Also, there is a need to evaluate the potential of IL-1β as atherapeutic drug target to decrease elevated levels of IL-1β as atreatment for IL-1β associated disease. This will present a need todetect velocity of decreasing IL-1β levels to evaluate effectiveness anddosing of therapies. This may prevent adverse events like Neutropeniathat develop after co-administration of drugs targeted to inflammatorycytokine pathways, like Kineret (IL-1Ra antagonist) and enteracept(TNF-alpha antagonist). To meet these goals, it is essential to have anassay that can detect IL-1β to below normal levels in human plasma.

The present invention provides an IL-1β assay that increases the utilityof IL-1β by allowing quantification at very low levels anddifferentiation between small changes in concentration that can provideinsights into drug efficacy or disease progression. See Example 24. TheIL-1β assay is sensitive enough to quantify IL-1β concentration inplasma from healthy, normal human subjects with previously unattainablelevels of accuracy and precision. The IL-1β assay enables the accuratequantification of IL-1β in human plasma with a broad dynamic range. Invarious embodiments, this assay will allow investigators to: (1) measurethe efficacy and dosing of therapeutics designed interfere with the IL-1mediated inflammatory response, such as Kineret; (2) design more robustclinical and preclinical studies when IL-1β concentration can be used asa therapeutic endpoint, as in the clinical trial of AMG 108; and (3)understand how IL-1β levels change in patients as they transition from ahealthy to diseased state.

In some embodiments, the method is capable of detecting the at a limitof detection of less than about 100, 80, 60, 50, 30, 20, 10, 5, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less thanabout 100 pg/ml. In some embodiments, the method is capable of detectingthe IL-1β at a limit of detection of less than about 200 pg/ml. In someembodiments, the method is capable of detecting the IL-1β at a limit ofdetection of less than about 150 pg/ml. In some embodiments, the methodis capable of detecting the IL-1β at a limit of detection of less thanabout 100 pg/ml. In some embodiments, the method is capable of detectingthe IL-1β a limit of detection of less than about 80 pg/ml. In someembodiments, the method is capable of detecting the IL-1β a limit ofdetection of less than about 60 pg/ml. In some embodiments, the methodis capable of detecting the IL-1β a limit of detection of less thanabout 50 pg/ml. In some embodiments, the method is capable of detectingthe IL-1β a limit of detection of less than about 30 pg/ml. In someembodiments, the method is capable of detecting the IL-1β a limit ofdetection of less than about 25 pg/ml. In some embodiments, the methodis capable of detecting the IL-1β a limit of detection of less thanabout 10 pg/ml. In some embodiments, the method is capable of detectingthe IL-1β a limit of detection of less than about 5 pg/ml. In someembodiments, the method is capable of detecting the IL-1β a limit ofdetection of less than about 1 pg/ml. In some embodiments, the method iscapable of detecting the IL-1β a limit of detection of less than about0.5 pg/ml. In some embodiments, the method is capable of detecting theIL-1β at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the IL-1β at a limit ofdetection of less than about 0.05 pg/ml. In some embodiments, the methodis capable of detecting the IL-1β at a limit of detection of less thanabout 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the IL-1β at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting the IL-1βat a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the IL-1β at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the IL-1β at a limit of detection of lessthan about 0.0001 pg/ml.

b. Interleukin 4 (IL-4)

Interleukin-4 (IL-4) is a cytokine that is a key regulator in humoraland adaptive immunity. IL-4 induces differentiation of naive helper Tcells (Th0 cells) to Th2 cells. It has many biological roles, includingthe stimulation of activated B-cell and T-cell proliferation, and thedifferentiation of CD4+ T-cells into Th2 cells IL-4 plays an importantrole in the development of allergic inflammatory responses. IL-4controls the production of IgE, expands IL-4 producing T cell subsetsand stabilizes effector cell functions.

IL-4 has therapeutic potential due to its role in the development ofallergic inflammatory responses. IL-4 also has shown to have promise indrug targeting for cancer. For example, PRX321 (Protox) is a targetedtherapeutic toxin in which IL-4 is linked to a Pseudomonas exo-toxin, apotent substance that can destroy cancer cells. Besides brain, kidneyand lung cancer, PRX321 has shown promising pre-clinical results in anumber of cancers over-expressing IL-4 receptors including pancreatic,ovarian, breast, head and neck, melanoma, prostate and blood cancerssuch as chronic lymphocytic leukemia (CLL) and Hodgkin's lymphoma.

The concentration of plasma IL-4 in healthy human subjects has yet to bedefined. Thus it is difficult to understand the role that differences inIL-4 concentrations play between disease and healthy states. Inaddition, measuring the efficacy of experimental therapeutics thattarget lowering IL-4 by measuring the velocity of IL-4 decreases ishindered by lack of assay sensitivity. Furthermore the reading range ofthe most sensitive ELISAs is limited to less than two logs, which forcessample retesting and wastage. Thus there is need for a highly sensitiveassay that can detect the velocity of subtle changes in concentration,and that can measure baseline concentration of IL-4 in normal subjects.

The IL-4 Assay provided by the present invention is sensitive enough toquantify IL-4 concentrations in plasma from healthy, normal humansubjects with a level of accuracy and precision currently unobtainableusing other high sensitivity assays. See Example 25. This assay enablesthe quantification of very low levels of plasma IL-4. In someembodiments, the assay allows the measurement of small changes in IL-4level that can provide insights into therapeutic efficacy. In variousembodiments, this assay allows investigators to: (1) measure theefficacy and dosing of therapeutics designed interfere with generalinflammatory and allergic responses; (2) design more robust clinical andpreclinical studies when IL-4 concentration is used as a therapeuticendpoint; and (3) understand how IL-4 levels change in patients as theytransition from a healthy to diseased state.

In some embodiments, the method of the present invention is capable ofdetecting IL-4 at a limit of detection of less than about 100, 80, 60,50, 30, 20, 10, 5, 1, 0.5, 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml,e.g., less than about 100 pg/ml. In some embodiments, the method iscapable of detecting the IL-4 at a limit of detection of less than about100 pg/ml. In some embodiments, the method is capable of detecting theIL-4 a limit of detection of less than about 80 pg/ml. In someembodiments, the method is capable of detecting the IL-4 a limit ofdetection of less than about 60 pg/ml. In some embodiments, the methodis capable of detecting the IL-4 a limit of detection of less than about50 pg/ml. In some embodiments, the method is capable of detecting theIL-4 a limit of detection of less than about 30 pg/ml. In someembodiments, the method is capable of detecting the IL-4 a limit ofdetection of less than about 25 pg/ml. In some embodiments, the methodis capable of detecting the IL-4 a limit of detection of less than about10 pg/ml. In some embodiments, the method is capable of detecting theIL-4 a limit of detection of less than about 5 pg/ml. In someembodiments, the method is capable of detecting the IL-4 a limit ofdetection of less than about 1 pg/ml. In some embodiments, the method iscapable of detecting the IL-4 a limit of detection of less than about0.5 pg/ml. In some embodiments, the method is capable of detecting theIL-4 at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the IL-4 at a limit ofdetection of less than about 0.05 pg/ml. IIn some embodiments, themethod is capable of detecting the IL-4 at a limit of detection of lessthan about 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the IL-4 at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting the IL-4at a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the IL-4 at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the IL4 at a limit of detection of lessthan about 0.0001 pg/ml.

c. Interleukin 6 (IL-6)

Interleukin-6 (IL-6) is a pro-inflammatory cytokine secreted by T cellsand macrophages to stimulate immune response to trauma, especially burnsor other tissue damage leading to inflammation. IL-6 is also secreted bymacrophages in response to specific microbial molecules, referred to aspathogen associated molecular patterns (PAMPs), which trigger the innateimmune response and initiate inflammatory cytokine production. IL-6 isone of the most important mediators of fever and of the acute phaseresponse. IL-6 is also called a “myokine,” a cytokine produced frommuscle, and is elevated in response to muscle contraction. Additionally,osteoblasts secrete IL-6 to stimulate osteoclast formation.

IL-6-related disorders include but are not limited to sepsis, peripheralarterial disease, and chronic obstructive pulmonary disease.Interleukin-6 mediated inflammation is also the common causative factorand therapeutic target for atherosclerotic vascular disease andage-related disorders including osteoporosis and type 2 diabetes. Inaddition, IL-6 can be measured in combination with other cytokines, forexample TNFα to diagnose additional diseases such as septic shock. IL-6has therapeutic potential as a drug target which would result in ananti-inflammatory and inhibition of the acute phase response. In termsof host response to a foreign pathogen, IL-6 has been shown, in mice, tobe required for resistance against the bacterium, Streptococcuspneumoniae. Inhibitors of IL-6 (including estrogen) are used to treatpostmenopausal osteoporosis. There is also therapeutic potential forcancer, as IL-6 is essential for hybridoma growth and is found in manysupplemental cloning media such as briclone.

Circulating levels of IL-6 in the plasma of healthy subjects isdifficult to determine with many currently available assays, thus it isdifficult to differentiate disease from healthy states. Furthermore,when used as a therapeutic target, it is desirous to measure therapeuticefficacy by measuring IL-6 levels as they decrease below normal statelevels. This can not be achieved with assays currently available. AnIL-6 assay is currently available outside the U.S. for diagnostic use,and for research use only (RUO) in the U.S. and Japan.

The present invention provides an IL-6 assay that enables thequantification of very low levels of plasma IL-6 and allows for accuratemeasurement of small changes in its level due to disease processes ortherapeutic intervention. See Example 26. This high level of sensitivitycan provide insights into therapeutic efficacy. In various embodiments,this assay will allow investigators to: (1) measure the efficacy anddosing of therapeutics designed interfere with the inflammatoryresponse; (2) design more robust clinical and preclinical studies whenIL-6 concentration is used as a therapeutic endpoint; and (3) understandhow IL-6 levels change in patients as they transition from a healthy todiseased state.

In some embodiments, the present invention provides a method capable ofdetecting IL-6 at a limit of detection of less than about 100, 80, 60,50, 30, 20, 10, 5, 1, 0.5, 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml,e.g., less than about 100 pg/ml. In some embodiments, the method iscapable of detecting the IL-6 at a limit of detection of less than about100 pg/ml. In some embodiments, the method is capable of detecting theIL-6 a limit of detection of less than about 80 pg/ml. In someembodiments, the method is capable of detecting the IL-6 a limit ofdetection of less than about 60 pg/ml. In some embodiments, the methodis capable of detecting the IL-6 a limit of detection of less than about50 pg/ml. In some embodiments, the method is capable of detecting theIL-6 a limit of detection of less than about 30 pg/ml. In someembodiments, the method is capable of detecting the IL-6 a limit ofdetection of less than about 25 pg/ml. In some embodiments, the methodis capable of detecting the IL-6 a limit of detection of less than about10 pg/ml. In some embodiments, the method is capable of detecting theIL-6 a limit of detection of less than about 5 pg/ml. In someembodiments, the method is capable of detecting the IL-6 a limit ofdetection of less than about 1 pg/ml. In some embodiments, the method iscapable of detecting the IL-6 a limit of detection of less than about0.5 pg/ml. In some embodiments, the method is capable of detecting theIL-6 at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the IL-6 at a limit ofdetection of less than about 0.05 pg/ml. In some embodiments, the methodis capable of detecting the IL-6 at a limit of detection of less thanabout 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the IL-6 at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting the IL-6at a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the IL-6 at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the IL-6 at a limit of detection of lessthan about 0.0001 pg/ml.

d. Interleukin 8 (IL-8)

Like IL-6, the present invention provides an Interleukin 8 (IL-8) assaythat enables the quantification of very low levels of plasma IL-8 andallows for accurate measurement of small changes in its level due todisease processes or therapeutic intervention. See FIG. 17. In someembodiments, the present invention provides a method capable ofdetecting IL-8 at a limit of detection of less than about 100, 80, 60,50, 30, 20, 10, 5, 1, 0.5, 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml,e.g., less than about 100 pg/ml. In some embodiments, the method iscapable of detecting the IL-8 at a limit of detection of less than about100 pg/ml. In some embodiments, the method is capable of detecting theIL-8 a limit of detection of less than about 80 pg/ml. In someembodiments, the method is capable of detecting the IL-8 a limit ofdetection of less than about 60 pg/ml. In some embodiments, the methodis capable of detecting the IL-8 a limit of detection of less than about50 pg/ml. In some embodiments, the method is capable of detecting theIL-8 a limit of detection of less than about 30 pg/ml. In someembodiments, the method is capable of detecting the IL-8 a limit ofdetection of less than about 25 pg/ml. In some embodiments, the methodis capable of detecting the IL-8 a limit of detection of less than about10 pg/ml. In some embodiments, the method is capable of detecting theIL-8 a limit of detection of less than about 5 pg/ml. In someembodiments, the method is capable of detecting the IL-8 a limit ofdetection of less than about 1 pg/ml. In some embodiments, the method iscapable of detecting the IL-8 a limit of detection of less than about0.5 pg/ml. In some embodiments, the method is capable of detecting theIL-8 at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the IL-8 at a limit ofdetection of less than about 0.05 pg/ml. In some embodiments, the methodis capable of detecting the IL-8 at a limit of detection of less thanabout 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the IL-8 at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting the IL-8at a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the IL-8 at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the IL-8 at a limit of detection of lessthan about 0.0001 pg/ml.

4. Inflammatory markers

Other cytokines that can be useful in detecting early onset ofinflammatory disease include markers and panels of markers ofinflammation as described herein. Examples of cytokines that can be usedto detect inflammatory disorders are Leukotriene 4 (LTE4), which can bean early marker of asthma, and TGFβ, which can be used to detect andmonitor the status of inflammatory disorders including fibrosis,sclerosis. Some markers can be used to detect more than one disorder,e.g., TGFβ, can also be used to detect the presence of cancer.

a. Leukotriene E4

Cysteinyl leukotrienes (LTC4, LTD4, LTE4) play an important role in thepathogenesis of asthma.

Leukotrienes are produced by mast cells, eosinophils, and other airwayinflammatory cells and increase vascular permeability, constrictbronchial smooth muscle, and mediate bronchial hyperresponsiveness.Levels of urinary LTE4, the stable metabolite of LTC4 and LTD4, areincreased in children and adults with asthma compared with healthycontrols and in asthmatics after bronchial challenge with antigen, afteroral challenge with aspirin in aspirin sensitive asthmatic subjects, andduring exercise induced bronchospasm. The importance of leukotrienes inthe pathology of asthma has been further demonstrated in large clinicaltrials with agents that block the actions of leukotrienes. For example,montelukast, a potent leukotriene receptor antagonist taken orally oncedaily, significantly improves asthma control in both children (aged 2-14years) and adults and attenuates exercise induced bronchoconstriction.

Activation of the leukotriene pathways is accompanied by rises inurinary levels of LTE4, and acute exacerbations of asthma areaccompanied by increased levels of LTE4 in urine followed by asignificant decrease during resolution. The degree of airflow limitationcorrelates with levels of urinary LTE4 during the exacerbation andfollow up periods, thus indicating that the leukotriene pathway isactivated during acute asthma. In addition, inhalation ofbronchoconstricting doses of LTC4 or LTE4 alter urinary LTE4 excretionin a dose-dependent manner thus indicating that urinary LTE4 can be usedas a marker of sulphidopeptide leukotriene synthesis in the lungs ofpatients with asthma.

The methods of the invention can be used to detect changes in LTE4 inbiological samples such as urinary samples. See Example 5. Measurementsof subnanogram levels of LTE4 can be useful as a reference for detectingand monitoring sulphidopeptide leukotriene synthesis in the lungs ofpatients with chronic or acute asthma.

In some embodiments, the methods of the present invention are capable ofdetecting LTE4 at a limit of detection of less than about 100, 80, 60,50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or0.0001 pg/ml, e.g., less than about 100 pg/ml. In some embodiments, themethod is capable of detecting LTE4 at a limit of detection of less thanabout 100 pg/ml. In some embodiments, the method is capable of detectingthe LTE4 a limit of detection of less than about 80 pg/ml. In someembodiments, the method is capable of detecting the LTE4 a limit ofdetection of less than about 60 pg/ml. In some embodiments, the methodis capable of detecting the LTE4 a limit of detection of less than about50 pg/ml. In some embodiments, the method is capable of detecting theLTE4 a limit of detection of less than about 30 pg/ml. In someembodiments, the method is capable of detecting the LTE4 a limit ofdetection of less than about 25 pg/ml. In some embodiments, the methodis capable of detecting the LTE4 a limit of detection of less than about10 pg/ml. In some embodiments, the method is capable of detecting theLTE4 a limit of detection of less than about 5 pg/ml. In someembodiments, the method is capable of detecting the LTE4 a limit ofdetection of less than about 1 pg/ml. In some embodiments, the method iscapable of detecting the LTE4 a limit of detection of less than about0.5 pg/ml. In some embodiments, the method is capable of detecting theLTE4 at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the LTE4 at a limit ofdetection of less than about 0.05 pg/ml. In some embodiments, the methodis capable of detecting the LTE4 at a limit of detection of less thanabout 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the LTE4 at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting the LTE4at a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the LTE4 at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the LTE4 at a limit of detection of lessthan about 0.0001 pg/ml.

b. TGFβ

The methods of the invention can also be performed to detect the earlyonset of diseases for which TGFβ is a marker. Examples of TGFβ-relateddiseases include fibrotic diseases. Fibrosis refers to the excessive andpersistent formation of scar tissue, which is responsible for morbidityand mortality associated with organ failure in a variety of chronicdiseases affecting the lungs, kidneys, eyes, heart, liver, and skin.TGFβ is well known for its role as a mediator of chronic fibroticeffects. For example, TGFβ is implicated in promoting fibroblasticproliferation and matrix accumulation in fibrotic lung disease.Inhibition of TGFβ has been proposed as a potential therapeutic avenuefor the management of lung fibrosis. TGFβ not only stimulates thesynthesis of many extracellular matrix molecules, including fibronectinand type I collagen and their receptors, but also decreases matrixdegradation via differential effects on the expression of proteases andtheir inhibitors, strongly promoting generation of extracellular matrix.Thus the analyzer systems of the invention can detect abnormal levels ofTGFβ, e.g., associated with fibrotic diseases, including but not limitedto idiopathic pulmonary fibrosis, diabetic nephropathy, progressivenephropathies including glomerulosclerosis and IgA nephropathy (causesof kidney failure and the need for dialysis and retransplant); diabeticretinopathy and advanced macular degeneration (fibrotic diseases of theeye and leading causes of blindness); cirrhosis and biliary atresia(leading causes of liver fibrosis and failure); congestive heartfailure; myocardiopathy associated with progressive fibrosis in Chagasdisease; lung fibrosis; and scleroderma.

TGFβ is also a marker for cancers including prostate cancer, cervicalcancer, lung carcinoma, and Hodgkin's disease. Plasma levels of TGFβ inpatients with lung cancer are often elevated. It has been shown that inpatients with an elevated plasma TGF beta 1 level at diagnosis,monitoring this level may be useful in detecting both diseasepersistence and recurrence after radiotherapy.

Transforming growth factor-beta (TGFβ) is also a multipotent growthfactor affecting development, homeostasis, and tissue repair. Increasedexpression of TGFβ has been reported in different malignancies,suggesting a role for this growth factor in tumorigenesis. Inparticular, it has been demonstrated that the presence of TGFβ in theendothelial and perivascular layers of small vessels in the tumor stromaof osteosarcomas suggests an angiogenic activity of this growth factor,and that increased expression of TGF-beta isoforms have been suggestedto play a role in the progression of osteosarcoma (Kloen et al., Cancer,80:2230-39 (1997)). TGFβ is one of the few known proteins that caninhibit cell growth. However, although the notion is controversial, someresearchers believe that some human malignancies such as breast cancersubvert TGFβ for their own purposes. In a paradox that is notunderstood, these cancers make TGFβ and steadily increase its expressionuntil it becomes a marker of advancing metastasis and decreasedsurvival. For example, levels of plasma TGFβ are markedly elevated inmen with prostate cancer metastatic to regional lymph nodes and bone. Inmen without clinical or pathologic evidence of metastases, thepreoperative plasma TGF-β level is a strong predictor of biochemicalprogression after surgery, presumably because of an association withoccult metastatic disease present at the time of radical prostatectomy.

In some embodiments, the method is capable of detecting TGF-β at a limitof detection of less than about 100, 80, 60, 50, 30, 20, 10, 5, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less thanabout 100 pg/ml. In some embodiments, the method is capable of detectingthe TGF-β at a limit of detection of less than about 100 pg/ml. In someembodiments, the method is capable of detecting the TGF-B a limit ofdetection of less than about 80 pg/ml. In some embodiments, the methodis capable of detecting the TGF-β a limit of detection of less thanabout 60 pg/ml. In some embodiments, the method is capable of detectingthe TGF-β a limit of detection of less than about 50 pg/ml. In someembodiments, the method is capable of detecting the TGF-β a limit ofdetection of less than about 30 pg/ml. In some embodiments, the methodis capable of detecting the TGF-β a limit of detection of less thanabout 25 pg/ml. In some embodiments, the method is capable of detectingthe TGF-β a limit of detection of less than about 10 pg/ml. In someembodiments, the method is capable of detecting the TGF-β a limit ofdetection of less than about 5 pg/ml. In some embodiments, the method iscapable of detecting the TGF-β a limit of detection of less than about 1pg/ml. In some embodiments, the method is capable of detecting the TGF-βa limit of detection of less than about 0.5 pg/ml. In some embodiments,the method is capable of detecting the TGF-β at a limit of detection ofless than about 0.1 pg/ml. In some embodiments, the method is capable ofdetecting the TGF-β at a limit of detection of less than about 0.05pg/ml. In some embodiments, the method is capable of detecting the TGF-βat a limit of detection of less than about 0.01 pg/ml. In someembodiments, the method is capable of detecting the TGF-β at a limit ofdetection of less than about 0.005 pg/ml. In some embodiments, themethod is capable of detecting the TGF-β at a limit of detection of lessthan about 0.001 pg/ml. In some embodiments, the method is capable ofdetecting the TGF-β at a limit of detection of less than about 0.0005pg/ml. In some embodiments, the method is capable of detecting the TGF-βat a limit of detection of less than about 0.0001 pg/ml.

Other markers of abnormal cell growth that are detected by the methodsof the invention include Akt1, Fas ligand, VEGF, Aβ-40, Aβ-42, cTnI,IL-1α, IL-1β, IL-4, and IL-6 as described herein.

5. Akt1

Akt1 is v-akt murine thymoma viral oncogene homolog 1 and is aserine-threonine protein kinase encoded by the AKT1 gene. Akt kinaseshave been implicated in disparate cell responses, including inhibitionof apoptosis and promotion of cell proliferation, angiogenesis, andtumor cell invasiveness.

Best known for its ability to inhibit apoptotic and non-apoptotic celldeath, Akt can be monitored to predict tumor response to anticancertreatment. Predicting tumor response by assessing the influence ofapoptosis and nonapoptotic cell death, would allow for developing a moreefficient strategy for enhancing the therapeutic effect of anticancertreatment. Anticancer treatment-induced apoptosis is regulated by thebalance of proapoptotic and antiapoptotic proteins through mitochondria,and resistance to apoptosis is mediated by Akt-dependent andBcl-2-dependent pathways. Bcl-2 partially inhibits nonapoptotic celldeath as well as apoptosis, whereas Akt inhibits both apoptotic andnonapoptotic cell death through several target proteins. Since drugsensitivity is likely correlated with the accumulation of apoptotic andnonapoptotic cell deaths, which may influence overall tumor response inanticancer treatment. The ability to predict overall tumor response fromthe modulation of several important cell death-related proteins mayresult in a more efficient strategy for improving the therapeuticeffect.

Akt1 is also involved in Epithelial-mesenchymal transition (EMT), whichis an important process during development and oncogenesis by whichepithelial cells acquire fibroblast-like properties and show reducedintercellular adhesion and increased motility. AKT is activated in manyhuman carcinomas, and the AKT-driven EMT may confer the motilityrequired for tissue invasion and metastasis. Thus future therapies basedon AKT inhibition may complement conventional treatments by controllingtumor cell invasion and metastasis. Akt is constitutively activated inmost melanoma cell lines and tumor samples of different progressionstages, and activation of AKT has been linked to the expression ofinvasion/metastasis-related melanoma cell adhesion molecule (MelCAM),which in turn is strongly associated with the acquisition of malignancyby human melanoma. Akt1 is also activated in pancreatic cancer, and AKTactivation has been shown to correlate with higher histologic tumorgrade. Thus, AKT activation is associated with tumor grade, an importantprognostic factor. Akt1 is also upregulated in prostate cancer and thatexpression is correlated with tumor progression. Thus, Akt1 could betargeted for therapeutic intervention of cancer while at its earlieststages. In some embodiments, the analyzer systems of the inventionprovide a method for providing an early diagnosis of a cancer bydetermining the presence or concentration of Akt1 in a sample from apatient when the level of Akt1 is less than about 100, 50, or 25 pg/ml.See Example 6.

In some embodiments, the methods of the present invention are capable ofdetecting Akt1 at a limit of detection of less than about 100, 80, 60,50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or0.0001 pg/ml, e.g., less than about 100 pg/ml. In some embodiments, themethod is capable of detecting Akt1 at a limit of detection of less thanabout 100 pg/ml. In some embodiments, the method is capable of detectingthe Akt1 a limit of detection of less than about 80 pg/ml. In someembodiments, the method is capable of detecting the Akt1 a limit ofdetection of less than about 60 pg/ml. In some embodiments, the methodis capable of detecting the Akt1 a limit of detection of less than about50 pg/ml. In some embodiments, the method is capable of detecting theAkt1 a limit of detection of less than about 30 pg/ml. In someembodiments, the method is capable of detecting the Akt1 a limit ofdetection of less than about 25 pg/ml. In some embodiments, the methodis capable of detecting the Akt1 a limit of detection of less than about10 pg/ml. In some embodiments, the method is capable of detecting theAkt1 a limit of detection of less than about 5 pg/ml. In someembodiments, the method is capable of detecting the Akt1 a limit ofdetection of less than about 1 pg/ml. In some embodiments, the method iscapable of detecting the Akt1 a limit of detection of less than about0.5 pg/ml. In some embodiments, the method is capable of detecting theAkt1 at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the Akt1 at a limit ofdetection of less than about 0.05 pg/ml. In some embodiments, the methodis capable of detecting the Akt1 at a limit of detection of less thanabout 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the Akt1 at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting the Akt1at a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the Akt1 at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the Akt1 at a limit of detection of lessthan about 0.0001 pg/ml.

6. Fas ligand

Fas Ligand (FasL), also known as CD95L, is a member of the TNF familyand induces apoptosis via binding to Fas (CD95). The protein exists intwo forms; either membrane FasL or soluble FasL, which migrate atmolecular weight of 45 kDa and 26 kDa, respectively. FasL is expressedon a variety of cells including activated lymphocytes, natural killercells and monocytes. Interaction of FasL and Fas plays an important rolein physiological apoptotic processes. Malfunction of the Fas-FasL systemcauses hyperplasia in peripheral lymphoid organs and acceleratesautoimmune disease progression and tumorigenesis. There are limited dataabout the levels of soluble apoptotic factors in general, and morespecifically about their modulation with therapeutic regimens.

The systems and methods of the invention can detect concentrations ofFas ligand that are as low as 2.4 pg/ml. Thus, in some embodiments, theanalyzer systems and methods of the invention provide for the detectionof Fas ligand to identify pathological conditions such as abnormallevels of apoptosis. Measurements of Fas in patient samples can be usedto diagnose conditions such as polycystic ovarian syndrome, tumors suchas testicular germ cell tumors, bladder cancer, lung cancer, and raretumors such as follicular dendritic cell tumors. In addition, Fasmeasurements of Fas ligand can be used to diagnose allograft rejectionand degenerative disease such as osteoarthritis. Thus, in someembodiments, the analyzer systems and methods of the invention can beused to determine the concentration of Fas ligand in a sample from apatient suspected of suffering from Fas ligand related disorder todiagnose the disorder, or the concentration of Fas ligand can be used tomonitor the progress or status of a Fas ligand related disorder in apatient undergoing therapy for the disorder. In some embodiments, theassay is capable of determining the level of Fas ligand in the sample ata concentration less than about 100, 50, 25, 10, or 5 pg/ml. See Example8.

In some embodiments, the methods of the present invention are capable ofdetecting FasL at a limit of detection of less than about 100, 80, 60,50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or0.0001 pg/ml, e.g., less than about 100 pg/ml. In some embodiments, themethod is capable of detecting FasL at a limit of detection of less thanabout 100 pg/ml. In some embodiments, the method is capable of detectingthe FasL a limit of detection of less than about 80 pg/ml. In someembodiments, the method is capable of detecting the FasL a limit ofdetection of less than about 60 pg/ml. In some embodiments, the methodis capable of detecting the FasL a limit of detection of less than about50 pg/ml. In some embodiments, the method is capable of detecting theFasL a limit of detection of less than about 30 pg/ml. In someembodiments, the method is capable of detecting the FasL a limit ofdetection of less than about 25 pg/ml. In some embodiments, the methodis capable of detecting the FasL a limit of detection of less than about10 pg/ml. In some embodiments, the method is capable of detecting theFasL a limit of detection of less than about 5 pg/ml. In someembodiments, the method is capable of detecting the FasL a limit ofdetection of less than about 1 pg/ml. In some embodiments, the method iscapable of detecting the FasL a limit of detection of less than about0.5 pg/ml. In some embodiments, the method is capable of detecting theFasL at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the FasL at a limit ofdetection of less than about 0.05 pg/ml. In some embodiments, the methodis capable of detecting the FasL at a limit of detection of less thanabout 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the FasL at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting the FasLat a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the FasL at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the FasL at a limit of detection of lessthan about 0.0001 pg/ml.

7. VEGF

Vascular endothelial growth factor-A (VEGF-A), commonly known as VEGF,is a member of a family of secreted glycoproteins that promoteendothelial cell growth, survival, migration, and vascular permeability,all of which contribute to angiogenesis. The binding of VEGF to itsreceptor triggers the activation of a cell signaling pathway that iscritical for the growth of blood vessels from pre-existing vasculature.VEGF is implicated in a variety of diseases including cancer,age-related macular degeneration, diabetic retinopathy and rheumatoidarthritis. As such, it is an attractive candidate for the development oftherapies to these diseases, particularly cancer.

The first anti-VEGF drug, the monoclonal antibody Avastin, was approvedby the FDA in 2004 and is approved to treat metastatic colon and nonsmall-cell lung cancer. The drug is also under study for the treatmentof many other cancers. Other compounds that target VEGF-mediated cellsignaling include the monoclonal antibody fragment Lucentis, approved totreat age related macular degeneration, and two small molecules, Sutentand Nexavar, which target receptor tyrosine kinases, including the VEGFreceptor. Other drug candidates targeting this path are in development.

With the efficacy seen with drugs that target VEGF and its pathway, VEGFis an attractive development target. In addition, as researchers studyvarious cancers and other diseases where VEGF signaling is implicated,measuring small changes in VEGF levels will help them understandbiological changes that occur as disease progresses. However, currentcommercially available immunoassays can only measure elevatedconcentrations of VEGF. They are not sensitive enough to measure VEGF inplasma obtained from healthy human subjects or detect the small changesin VEGF levels that may be indicative of an early disease state.However, the plasma VEGF assay according to the present inventionprovides the power needed to use VEGF as a biomarker for disease and thesensitivity to quantify VEGF in healthy human subjects as well as thoseundergoing anti-VEGF therapy. In some embodiments, the human VEGF assayhas an LOD of about 0.1 pg/ml and a lower limit of quantitation (LLOQ)of 0.3 pg/ml, making it 90× more sensitive than the commonly used ELISAassay. See Examples 11-21.

The present invention increases the clinical utility of VEGF by allowingscientists to detect very low levels of VEGF and measure small changesin its level that can provide insights into drug efficacy or diseaseprogression. Among other improvements, the assay allows investigatorsto: (1) measure the efficacy and dosing of therapeutics designed tolower the levels of VEGF, particularly when VEGF levels should go muchlower than that seen in normal states; (2) design more robust clinicaland preclinical studies when VEGF concentration is used as a therapeuticendpoint; and (3) understand how VEGF levels change in patients as theytransition from a healthy to diseased state with cancer and otherdiseases involving angiogenesis.

In some embodiments, the present invention provides methods to quantifynormal levels of VEGF, and identify abnormally elevated levels of VEGFindicative of the presence of an early stage cancer/tumor. Typicalhealthy levels of VEGF in humans are less than 50 pg/mL, and aresignificantly elevated (>100 pg/mL, often 200-500 pg/mL) in subjectswith cancer. In other embodiments, the methods described herein can beused to indicate the presence of other cancers, such as prostate andlymphoma. The method can be used to indicate the presence of solidtumors that are undergoing vascularization, which will have increasedlevels of VEGF.

In some embodiments, the present invention provides methods to quantifynormal levels of VEGF, and identify abnormally elevated levels of VEGFindicative of the presence of vascular inflammation. This measurementcan be augmented by co-measurement of other inflammatory cytokines inhealthy individuals, where elevated levels are indicative ofinflammation. In some embodiments, because of the role of VEGF inangiogenesis and artherosclerosis, the invention can also be used toquantify abnormally elevated levels of VEGF as indicative of cardiacdisease in conjunction with elevated levels of cTnI which is the goldstandard for detecting myocardial infarction. This measurement can beaugmented by co-measurement of other cardiac markers (i.e., pro-BNP) orinflammatory markers (i.e., hsCRP, cytokines) in healthy individuals,where elevated levels are indicative of cardiac disease. In someembodiments, the method described can be used to quantify normal levelsof VEGF, and identify abnormally elevated levels of VEGF indicative ofthe presence of artherosclerosis in subjects with diabetes. Thismeasurement can be augmented by co-measurement of other markers fordiabetes (i.e., insulin) and for metabolic disease (i.e., glucagon likepeptide-1 (GLP-1)).

The present invention provides methods to measure VEGF in very smallsample volumes that are less than the standard sample volume of 100 μl.The methods are enabled by the sensitivity of the assay and enable agreater number of samples to provide quantifiable results in smallvolume samples compared to other methods. In one embodiment, the methodsmeasure VEGF in human or mouse plasma samples of less than or equal to10 μl. In another embodiment, the methods measure VEGF in tissue lysatesfrom human or mouse plasma samples of less than or equal to 10 μl. Thesemethods have been tested in lysates from human breast cancer tissuebiopsies, as well as in mouse tissue lysates from several strains ofmice. In another embodiment, the methods measure VEGF in lysatesprepared from tissue biopsies in healthy and diseased individuals. Basedon a typical 1 mm needle biopsy, and resulting lysates volume of lessthan or equal to 10 μl, this method enables quantification of VEGF froma needle biopsy. Small volume sample sizes are also provided with othermarkers of the present invention.

In one aspect, the present invention provides a method for determiningthe presence or absence of a single molecule of VEGF or a fragment orcomplex thereof in a sample, by i) labeling the molecule, fragment, orcomplex, if present, with a label; and ii) detecting the presence orabsence of the label, where the detection of the presence of the labelindicates the presence of the single molecule, fragment, or complex ofVEGF in the sample. In some embodiments, the methods of the presentinvention are capable of detecting VEGF at a limit of detection of lessthan about 115, 100, 80, 60, 50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01,0.005, 0.001, 0.0005 or 0.0001 pg/ml, e.g., less than about 115 pg/ml.In some embodiments, the method is capable of detecting VEGF at a limitof detection of less than about 115 pg/ml. In some embodiments, themethod is capable of detecting VEGF at a limit of detection of less thanabout 100 pg/ml. In some embodiments, the method is capable of detectingthe VEGF a limit of detection of less than about 80 pg/ml. In someembodiments, the method is capable of detecting the VEGF a limit ofdetection of less than about 60 pg/ml. In some embodiments, the methodis capable of detecting the VEGF a limit of detection of less than about50 pg/ml. In some embodiments, the method is capable of detecting theVEGF a limit of detection of less than about 30 pg/ml. In someembodiments, the method is capable of detecting the VEGF a limit ofdetection of less than about 25 pg/ml. In some embodiments, the methodis capable of detecting the VEGF a limit of detection of less than about10 pg/ml. In some embodiments, the method is capable of detecting theVEGF a limit of detection of less than about 5 pg/ml. In someembodiments, the method is capable of detecting the VEGF a limit ofdetection of less than about 1 pg/ml. In some embodiments, the method iscapable of detecting the VEGF a limit of detection of less than about0.5 pg/ml. In some embodiments, the method is capable of detecting theVEGF at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the VEGF at a limit ofdetection of less than about 0.05 pg/ml. In some embodiments, the methodis capable of detecting the VEGF at a limit of detection of less thanabout 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the VEGF at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting the VEGFat a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the VEGF at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the VEGF at a limit of detection of lessthan about 0.0001 pg/ml.

8. Aβ-40 and Aβ-42

Amyloid beta proteins (40 and 42 amino acids) are the main constituentof amyloid plaques in the brains of Alzheimer's disease (AD) patients.In healthy and diseased states Aβ-40 is the more common form (10-20×higher than Aβ-42) of the two in both cerebrospinal fluid (CSF) andplasma. In patients with AD, Aβ-42 primarily aggregates and deposits inthe brain forming plaques. Thus the concentration of Aβ-42 is decreasedin the CSF of many AD patients. Recent studies suggest that a decreasein Aβ-42 concentrations (with a paralleled change in the ratio ofAβ-40/Aβ-42) in CSF and plasma are predictive of the onset of AD.

There is no cure for Alzheimer's disease and currently availabletherapeutics minimize some of the symptoms associated with AD but do notslow disease progression. Numerous experimental approaches focus onminimizing Aβ-42 levels by preventing production of or lowering Aβ-42concentrations, stimulating the immune system to attack Aβ proteins aswell as preventing Aβ proteins from aggregating and forming plaques. Animportant component in designing therapeutic trials is to identifypatients that are at risk for developing AD such that studies can beperformed in a cost effective timely manner. Hence biomarkers would beinvaluable for both understanding Aβ levels as surrogate endpoints aswell as in efficient study design.

Preventive therapy is a major focus as the best way to manage AD.Guidelines describe the need for non-invasive biomarkers that can beused to predict and diagnose the formation of AD. Such information willbe invaluable for clinical study design, as well as the evaluation oftherapeutic effectiveness. Measuring Aβ-40 and Aβ-42 concentrations inplasma provide promise for such information. In healthy normal humans,plasma concentrations range from 200-400 pg/ml (Aβ-40) and 15-30 pg/ml(Aβ-42). However with AD, Aβ-42 levels decrease, and are oftenundetectable by currently available EIA technology. Furthermore,interventional strategies based on depleting Aβ-42 formation requiremethods that measure decreases in Aβ-42. Thus there is a need toaccurately and precisely quantify low concentrations of amyloid proteinsin plasma.

The Aβ-40 and Aβ-42 assays according to the present invention allow thequantification of amyloid beta proteins from human plasma withexceptional sensitivity, enabling the use of Aβ-40/Aβ-42 as a velocitybiomarker in Alzheimer's disease studies and to evaluate therapeuticinterventions. See Example 22. Among other advantages, this assay allowsinvestigators to: (1) identify subjects with potential high risk fordeveloping AD and hence design interventional studies that include highrisk for disease development; (2) design more robust clinical andpreclinical studies when Aβ protein concentrations are used as atherapeutic endpoint; and (3) understand how Aβ protein levels change inhumans as they transition from a healthy to a diseased state.

In some embodiments, the methods of the present invention are capable ofdetecting the Aβ-40 at a limit of detection of less than about 100, 80,60, 50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or0.0001 pg/ml, e.g., less than about 100 pg/ml. In some embodiments, themethod is capable of detecting Aβ-40 at a limit of detection of lessthan about 100 pg/ml. In some embodiments, the method is capable ofdetecting the Aβ-40 a limit of detection of less than about 80 pg/ml. Insome embodiments, the method is capable of detecting the Aβ-40 a limitof detection of less than about 60 pg/ml. In some embodiments, themethod is capable of detecting the Aβ-40 a limit of detection of lessthan about 50 pg/ml. In some embodiments, the method is capable ofdetecting the Aβ-40 a limit of detection of less than about 30 pg/ml. Insome embodiments, the method is capable of detecting the Aβ-40 a limitof detection of less than about 25 pg/ml. In some embodiments, themethod is capable of detecting the Aβ-40 a limit of detection of lessthan about 10 pg/ml. In some embodiments, the method is capable ofdetecting the Aβ-40 a limit of detection of less than about 5 pg/ml. Insome embodiments, the method is capable of detecting the Aβ-40 a limitof detection of less than about 1 pg/ml. In some embodiments, the methodis capable of detecting the Aβ-40 a limit of detection of less thanabout 0.5 pg/ml. In some embodiments, the method is capable of detectingthe Aβ-40 at a limit of detection of less than about 0.1 pg/ml. In someembodiments, the method is capable of detecting the Aβ-40 at a limit ofdetection of less than about 0.05 pg/ml. In some embodiments, the methodis capable of detecting the Aβ-40 at a limit of detection of less thanabout 0.01 pg/ml. In some embodiments, the method is capable ofdetecting the Aβ-40 at a limit of detection of less than about 0.005pg/ml. In some embodiments, the method is capable of detecting the Aβ-40at a limit of detection of less than about 0.001 pg/ml. In someembodiments, the method is capable of detecting the Aβ-40 at a limit ofdetection of less than about 0.0005 pg/ml. In some embodiments, themethod is capable of detecting the Aβ-40 at a limit of detection of lessthan about 0.0001 pg/ml.

In some embodiments, the method is capable of detecting the Aβ-42 at alimit of detection of less than about 250, 200, 150, 100, 80, 60, 50,30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 or 0.0001pg/ml, e.g., less than about 200 pg/ml. In some embodiments, the methodis capable of detecting Aβ-42 at a limit of detection of less than about200 pg/ml. In some embodiments, the method is capable of detecting Aβ-42at a limit of detection of less than about 150 pg/ml. In someembodiments, the method is capable of detecting Aβ-42 at a limit ofdetection of less than about 100 pg/ml. In some embodiments, the methodis capable of detecting the Aβ-42 a limit of detection of less thanabout 80 pg/ml. In some embodiments, the method is capable of detectingthe Aβ-42 a limit of detection of less than about 60 pg/ml. In someembodiments, the method is capable of detecting the Aβ-42 a limit ofdetection of less than about 50 pg/ml. In some embodiments, the methodis capable of detecting the Aβ-42 a limit of detection of less thanabout 30 pg/ml. In some embodiments, the method is capable of detectingthe Aβ-42 a limit of detection of less than about 25 pg/ml. In someembodiments, the method is capable of detecting the Aβ-42 a limit ofdetection of less than about 10 pg/ml. In some embodiments, the methodis capable of detecting the Aβ-42 a limit of detection of less thanabout 5 pg/ml. In some embodiments, the method is capable of detectingthe Aβ-42 a limit of detection of less than about 1 pg/ml. In someembodiments, the method is capable of detecting the Aβ-42 a limit ofdetection of less than about 0.5 pg/ml. In some embodiments, the methodis capable of detecting the Aβ-42 at a limit of detection of less thanabout 0.1 pg/ml. In some embodiments, the method is capable of detectingthe Aβ-42 at a limit of detection of less than about 0.05 pg/ml. In someembodiments, the method is capable of detecting the Aβ-42 at a limit ofdetection of less than about 0.01 pg/ml. In some embodiments, the methodis capable of detecting the Aβ-42 at a limit of detection of less thanabout 0.005 pg/ml. In some embodiments, the method is capable ofdetecting the Aβ-42 at a limit of detection of less than about 0.001pg/ml. In some embodiments, the method is capable of detecting the Aβ-42at a limit of detection of less than about 0.0005 pg/ml. In someembodiments, the method is capable of detecting the Aβ-42 at a limit ofdetection of less than about 0.0001 pg/ml.

C. Multiple Marker Panels

Medical diagnostics have traditionally relied upon the detection ofsingle molecular markers (e.g., gene mutations, elevated PSA levels).Unfortunately, single markers approaches are suboptimal to detect ordifferentiate many biological states or diseases, e.g., cancer. Thus, insome cases, assays that recognize only a single marker have limitedpredictive value. According to the methods of the present invention, thescreening, diagnosis, and therapeutic monitoring of such biologicalstates, e.g., diseases, using a plurality of markers can providesignificant improvements over methods that use single marker analyses.This multiplexed approach is particularly well suited for cancerdiagnostics because cancer is a highly complex disease, thismulti-factorial “panel” approach is consistent with the heterogeneousnature of cancer, both cytologically and clinically.

Key to the successful implementation of a panel approach to medicaltests is the design and development of optimized panels of markers thatcan characterize and distinguish biological states. Two key evaluativemeasures of any medical screening or diagnostic test are its sensitivityand specificity, which measure how well the test performs to accuratelydetect all affected individuals without exception, and without falselyincluding individuals who do not have the target disease (predicitivevalue). Historically, many diagnostic tests have been criticized due topoor sensitivity and specificity.

A true positive (TP) result is where the test is positive and thecondition is present. A false positive (FP) result is where the test ispositive but the condition is not present. A true negative (TN) resultis where the test is negative and the condition is not present. A falsenegative (FN) result is where the test is negative but the condition ispresent. In this context: Sensitivity=TP/(TP+FN);Specificity=TN/(FP+TN); and Predictive value=TP/(TP+FP).

Sensitivity is a measure of a test's ability to correctly detect thetarget disease in an individual being tested. A test having poorsensitivity produces a high rate of false negatives, i.e., individualswho have the disease but are falsely identified as being free of thatparticular disease. The potential danger of a false negative is that thediseased individual will remain undiagnosed and untreated for someperiod of time, during which the disease may progress to a later stagewherein treatments, if any, may be less effective. An example of a testthat has low sensitivity is a protein-based blood test for HIV. Thistype of test exhibits poor sensitivity because it fails to detect thepresence of the virus until the disease is well established and thevirus has invaded the bloodstream in substantial numbers. In contrast,an example of a test that has high sensitivity is viral-load detectionusing the polymerase chain reaction (PCR). High sensitivity is achievedbecause this type of test can detect very small quantities of the virus.High sensitivity is particularly important when the consequences ofmissing a diagnosis are high.

Specificity, on the other hand, is a measure of a test's ability toidentify accurately patients who are free of the disease state. A testhaving poor specificity produces a high rate of false positives, i.e.,individuals who are falsely identified as having the disease. A drawbackof false positives is that they force patients to undergo unnecessarymedical procedures treatments with their attendant risks, emotional andfinancial stresses, and which could have adverse effects on thepatient's health. A feature of diseases which makes it difficult todevelop diagnostic tests with high specificity is that diseasemechanisms, particularly in cancer, often involve a plurality of genesand proteins. Additionally, certain proteins may be elevated for reasonsunrelated to a disease state. An example of a test that has highspecificity is a gene-based test that can detect a p53 mutation.Specificity is important when the cost or risk associated with furtherdiagnostic procedures or further medical intervention is very high.

Those of skill in the art will appreciate that statistical approacheshave been developed to combine the data from multiple marker and providea statistical likelihood of the presence of a biological state, e.g.,the presence of a disease such as cancer. Examples of such methods aredisclosed in U.S. patent application Ser. Nos. 11/934,008; 11/939,484;and 11/640,511. In one embodiment, the concentration of the panelmembers in a patient sample can be combined using a logisticalregression and the disease status of the subject can be determined usinga Receiver-Operating Characteristic (ROC) analysis. See, e.g., U.S.patent application Ser. Nos. 11/934,008; 11/939,484; and 11/640,511. Inother approaches, statistical methods can be used to classify the samplebased on the detection of the marker panels. E.g., the results of themarker assays can be used to classify a sample as diseased or healthy.Such classification (pattern recognition) methods include, e.g.,Bayesian classifiers, profile similarity, artificial neural networks,support vector machines (SVM), logistic or logic regression, linear orquadratic discriminant analysis, decision trees, clustering, principalcomponent analysis, Fischer's discriminate analysis or nearest neighborclassifer analysis. Machine learning approaches to classificationinclude, e.g., weighted voting, k-nearest neighbors, decision treeinduction, support vector machines (SVM), and feed-forward neuralnetworks. Such methods are known to those of skill in the art.

In other embodiments, simpler schemes can be used. For example, in oneembodiment, the elevated concentration of two markers may indicate thepresence of a biological state, e.g., a disease. In another embodiment,the decreasing concentration of two markers may indicate the presence ofa biological state, e.g., a disease. In another embodiment, an increasedconcentration of one marker and a decreased concentration of anothermarker may indicate the presence of a biological state, e.g., a disease.Using such methodology, the results of a second marker provide a medicalpractitioner with increased confidence in a diagnosis, prognosis, orcourse of treatment. The multiple markers can provide a confirmatorydetection, diagnosis, prognosis, or the like. It will be appreciatedthat any of the above methods can be used for three markers, fourmarkers, etc.

1. Multiple biomarker panels

The methods of the present invention described for quantitativemeasurement of biomarkers, e.g., cTnI, cytokines, or VEGF, can becombined with measurement of other biomarkers quantified utilizing thesame technology. See FIG. 4. These multiple marker assays can improvethe sensitivity and specificity of the detection and monitoring of acondition in a subject. Such assays remain highly sensitive and have thecapability to accurately quantify each analyte across a normal, healthyreference range. As disclosed herein, markers of the present inventioninclude, for example, any composition and/or molecule or a complex ofcompositions and/or molecules that is associated with a biological stateof an organism (e.g., a condition such as a disease or a non-diseasestate).

In one embodiment, the present invention provides a method to detect ormonitor a condition in a subject, comprising detecting a first marker ina first sample from the subject and detecting a second marker, whereinthe first marker comprises Cardiac Troponin-I (cTnI) or VascularEndothelial Growth Factor (VEGF), and wherein the limit of detection ofthe first marker is less than about 10 pg/ml. In some embodiments, thelimit of detection of the first marker is less than about 100 pg/ml. Insome embodiments, the limit of detection of the first marker is lessthan about 50 pg/ml. In some embodiments, the limit of detection of thefirst marker is less than about 5 pg/ml. In some embodiments, the limitof detection of the first marker is less than about 1 pg/ml. In someembodiments, the limit of detection of the first marker is less thanabout 0.5 pg/ml. In some embodiments, the limit of detection of thefirst marker is less than about 0.1 pg/ml. In some embodiments, thelimit of detection of the first marker is less than about 0.05 pg/ml. Insome embodiments, the limit of detection of the first marker is lessthan about 0.01 pg/ml. In some embodiments, the limit of detection ofthe first marker is less than about 0.005 pg/ml. In some embodiments,the limit of detection of the first marker is less than about 0.001pg/ml. In some embodiments, the limit of detection of the first markeris less than about 0.0005 pg/ml. In some embodiments, the limit ofdetection of the first marker is less than about 0.0001 pg/ml. In someembodiments, the limit of detection of the first marker ranges fromabout 10 pg/ml to about 0.01 pg/ml. In some embodiments, the limit ofdetection of the first marker ranges from about 5 pg/ml to about 0.01pg/ml. In some embodiments, the limit of detection of the first markerranges from about 1 pg/ml to about 0.01 pg/ml. In some embodiments, thelimit of detection of the first marker ranges from about 10 pg/ml toabout 0.001 pg/ml. In some embodiments, the limit of detection of thefirst marker ranges from about 5 pg/ml to about 0.001 pg/ml. In someembodiments, the limit of detection of the first marker ranges fromabout 1 pg/ml to about 0.001 pg/ml. In some embodiments, the limit ofdetection of the first marker ranges from about 10 pg/ml to about 0.0001pg/ml. In some embodiments, the limit of detection of the first markerranges from about 5 pg/ml to about 0.0001 pg/ml. In some embodiments,the limit of detection of the first marker ranges from about 1 pg/ml toabout 0.0001 pg/ml.

In some embodiments, the sample comprises plasma, serum, cell lysates orother samples as disclosed herein. For example, the present inventioncan be used to measure VEGF in the plasma of humans and mice, asdisclosed herein.

An advantage of the present invention is its robustness. The level ofreproducibility allows for more sensitive detection across a broad rangeof detection. The present invention provides advantages even when thelimit of detection is below the typical or expected level of a givenmarker because the variation at higher levels can be reduced. In someembodiments, the coefficient of variation (CV) of the limit of detectionranges from about 100% to about 1%. In some embodiments, the coefficientof variation (CV) of the limit of detection ranges from about 90% toabout 1%. In some embodiments, the coefficient of variation (CV) of thelimit of detection ranges from about 80% to about 1%. In someembodiments, the coefficient of variation (CV) of the limit of detectionranges from about 70% to about 1%. In some embodiments, the coefficientof variation (CV) of the limit of detection ranges from about 60% toabout 1%. In some embodiments, the coefficient of variation (CV) of thelimit of detection ranges from about 50% to about 1%. In someembodiments, the coefficient of variation (CV) of the limit of detectionranges from about 40% to about 1%. In some embodiments, the coefficientof variation (CV) of the limit of detection ranges from about 30% toabout 1%. In some embodiments, the coefficient of variation (CV) of thelimit of detection ranges from about 20% to about 1%. In someembodiments, the coefficient of variation (CV) of the limit of detectionranges from about 15% to about 1%. In some embodiments, the coefficientof variation (CV) of the limit of detection ranges from about 10% toabout 1%. In some embodiments, the coefficient of variation (CV) of thelimit of detection ranges from about 5% to about 1%.

Because of the sensitivity of the methods of the present invention, verysmall sample volumes can be used. For example, the methods here can beused to measure VEGF in small sample volumes, e.g., 10 μl or less,compared to the standard sample volume of 100 μl. The present inventionenables a greater number of samples to provide quantifiable results insmall volume samples compared to other methods. For example, a lysateprepared from a typical 1 mm needle biopsy may have a volume less thanor equal to 10 μl. Using the present invention, such sample can beassayed. In some embodiments, the present invention allows the use ofsample volume under 100 μl. In some embodiments, the present inventionallows the use of sample volume under 90 μl. In some embodiments, thepresent invention allows the use of sample volume under 80 μl. In someembodiments, the present invention allows the use of sample volume under70 μl. In some embodiments, the present invention allows the use ofsample volume under 60 μl. In some embodiments, the present inventionallows the use of sample volume under 50 μl. In some embodiments, thepresent invention allows the use of sample volume under 40 μl. In someembodiments, the present invention allows the use of sample volume under30 μl. In some embodiments, the present invention allows the use ofsample volume under 25 μl. In some embodiments, the present inventionallows the use of sample volume under 20 μl. In some embodiments, thepresent invention allows the use of sample volume under 15 μl. In someembodiments, the present invention allows the use of sample volume under10 μl. In some embodiments, the present invention allows the use ofsample volume under 5 μl. In some embodiments, the present inventionallows the use of sample volume under 1 μl. In some embodiments, thepresent invention allows the use of sample volume under 0.05 μl. In someembodiments, the present invention allows the use of sample volume under0.01 μl. In some embodiments, the present invention allows the use ofsample volume under 0.005 μl. In some embodiments, the present inventionallows the use of sample volume under 0.001 μl. In some embodiments, thepresent invention allows the use of sample volume under 0.0005 μl. Insome embodiments, the present invention allows the use of sample volumeunder 0.0001 μl. In some embodiments, the range of the sample size isabout 10 μl to about 0.1 μl. In some embodiments, the range of thesample size is about 10 μl to about 1 μl. In some embodiments, the rangeof the sample size is about 5 μl to about 1 μl. In some embodiments, therange of the sample size is about 5 μl to about 0.1 μl.

In some embodiments, the second marker comprises a biomarker, e.g., aprotein or a nucleic acid. As disclosed herein, when the first marker orthe second marker is a protein, this is understood to encompass afragment or complex of the protein, or a polypeptide. In embodimentswherein the second marker is such a protein, the limit of detection ofthe second marker can range from about 10 pg/ml to about 0.1 pg/ml. Insome embodiments, the limit of detection of the second marker is lessthan about 100 pg/ml. In some embodiments, the limit of detection of thesecond marker is less than about 10 pg/ml. In some embodiments, thelimit of detection of the second marker is less than about 5 pg/ml. Insome embodiments, the limit of detection of the second marker is lessthan about 1 pg/ml. In some embodiments, the limit of detection of thesecond marker is less than about 0.5 pg/ml. In some embodiments, thelimit of detection of the second marker is less than about 0.1 pg/ml. Insome embodiments, the limit of detection of the second marker is lessthan about 0.05 pg/ml. In some embodiments, the limit of detection ofthe second marker is less than about 0.01 pg/ml. In some embodiments,the limit of detection of the second marker is less than about 0.005pg/ml. In some embodiments, the limit of detection of the second markeris less than about 0.001 pg/ml. In some embodiments, the limit ofdetection of the second marker is less than about 0.0005 pg/ml. In someembodiments, the limit of detection of the second marker is less thanabout 0.0001 pg/ml. In some embodiments, the limit of detection of thesecond marker ranges from about 10 pg/ml to about 0.01 pg/ml. In someembodiments, the limit of detection of the second marker ranges fromabout 5 pg/ml to about 0.01 pg/ml. In some embodiments, the limit ofdetection of the second marker ranges from about 1 pg/ml to about 0.01pg/ml. In some embodiments, the limit of detection of the second markerranges from about 10 pg/ml to about 0.001 pg/ml. In some embodiments,the limit of detection of the second marker ranges from about 5 pg/ml toabout 0.001 pg/ml. In some embodiments, the limit of detection of thesecond marker ranges from about 1 pg/ml to about 0.001 pg/ml. In someembodiments, the limit of detection of the second marker ranges fromabout 10 pg/ml to about 0.0001 pg/ml. In some embodiments, the limit ofdetection of the second marker ranges from about 5 pg/ml to about 0.0001pg/ml. In some embodiments, the limit of detection of the second markerranges from about 1 pg/ml to about 0.0001 pg/ml.

The second marker can be any biomarker indicative of a biological state.Numerous such biomarkers are disclosed herein. The second marker may bemeasured by the methods of the present invention or may be measuredusing alternate, e.g., preexisting methods. In some embodiments, thesecond marker is detected using the methods of the present invention. Insome embodiments, the second marker is detected using commerciallyavailable kits from a variety of suppliers. These include commerciallyavailable kits which can be used to detect the second marker includeaffinity purified antibodies and conjugates, western blotting kits andreagents, recombinant protein detection and analysis, elisa kits andreagents, immunohistology kits and reagents, sample preparation andprotein purification, and protein labeling kits and reagents. Companiesproviding such kits include Invitrogen, Millipore, R&D Systems, CogentDiagnostics, Biihlmann Laboratories AG, Quidel, and Scimedx Corporation.Indeed, the methods of the present invention can be combined with anymethod to detect another biomarker.

In some embodiments, the second marker is a biomarker that comprisesproBNP, IL-1α, IL-1β, IL-6, IL-8, IL-10, TNF-α, IFN-γ, cTnI, VEGF,insulin, GLP-1, TREM1, Leukotriene E4, Akt1, Aβ-40, Aβ-42, or Fasligand. In some embodiments, the second marker is a cytokine. Asdisclosed herein, currently over 100 cytokines/chemokines whosecoordinate or discordant regulation is of clinical interest, any ofwhich can be detected with the methods of the invention. In someembodiments, the cytokine is G-CSF, MIP-1α, IL-10, IL-22, IL-8, IL-5,IL-21, INF-γ, IL-15, IL-6, TNF-α, IL-7, GM-CSF, IL-2, IL-4, IL-1α,IL-12, IL-17α, IL-1β, MCP, IL-32 or RANTES. In some embodiments, thecytokine is IL-10, IL-8, INF-γ, IL-6, TNF-α, IL-7, IL-1α, or IL-1β. Inother embodiments, the second marker is a high abundance protein. Insuch embodiments, the second marker can be an apolipoprotein,ischemia-modified albumin (IMA), fibronectin, C-reactive protein (CRP),B-type Natriuretic Peptide (which includes BNP, proBNP and NT-proBNP),or Myeloperoxidase (MPO).

In some embodiments, the methods provided comprise determining aconcentration for the first marker, i.e., cTnI or VEGF, and determininga concentration for the second marker if the second marker is abiomarker, e.g., a protein. In some embodiments, the methods providedcomprise determining a ratio of a concentration of the first markercompared to a concentration for the second marker. Methods to determinea concentration using the devices and methods of the present inventionare disclosed herein. Commercial kits, e.g., commercial ELISA kits, canalso be used to determine a protein concentration, e.g., by comparingthe level of the biomarker being detected against a standard curve.

2. Mixed marker panels

The methods of the present invention can also be combined with othertypes of markers which serve as a metric for a desired biological state,e.g., a disease state. See FIG. 4. Examples include physiologicalmarkers (stress testing, insulin tolerance, BMI, blood pressure, sleepapnea), molecular markers (cholesterol, LDL/HDL, vitamin-D), highabundance proteins (apolipoproteins, IMA, fibronectin), and geneticmarkers for disease. In some embodiments, the second marker is aphysiological marker. In some embodiments, the second marker is amolecular marker. In some embodiments, the second marker is a geneticmarker.

In one embodiment, the present invention provides a method to detect ormonitor a condition in a subject, comprising detecting a first marker ina first sample from the subject and detecting a second marker, whereinthe first marker comprises Cardiac Troponin-I (cTnI) or VascularEndothelial Growth Factor (VEGF) and the second marker comprises aphysiological marker. Examples of physiological markers include anelectrocardiogram (EKG), stress testing, nuclear imaging, ultrasound,insulin tolerance, body mass index, bone mass, blood pressure, age, sex,sleep apnea, medical history, or other physiological conditions. In oneembodiment, the second marker comprises a medical procedure fordetermining whether a subject has coronary artery disease or is at riskfor experiencing a complication of coronary artery disease include, butare not limited to, coronary angiography, coronary intravascularultrasound (IVUS), stress testing (with and without imaging), assessmentof carotid intimal medial thickening, carotid ultrasound studies with orwithout implementation of techniques of virtual histology, coronaryartery electron beam computer tomography (EBTC), cardiac computerizedtomography (CT) scan, CT angiography, cardiac magnetic resonance imaging(MRI), and magnetic resonance angiography (MRA). The present methods arealso useful for monitoring subjects at risk of having a cardiovasculardisease, wherein the second marker is a risk factor. Risk factors forcardiac diseases include elevated levels of circulating MPO,hypertension, family history of premature CVD, smoking, high totalcholesterol, low HDL cholesterol, obesity, diabetes, etc. Becausecardiovascular disease, typically, is not limited to one region of asubject's vasculature, a subject who is diagnosed as having or being atrisk of having coronary artery disease is also considered at risk ofdeveloping or having other forms of CVD such as cerebrovascular disease,aortic-iliac disease, and peripheral artery disease. Subjects who are atrisk of having cardiovascular disease are at risk of having an abnormalstress test or abnormal cardiac catherization. Subjects who are at riskof having CVD are also at risk of exhibiting increased carotid intimalmedial thickness and coronary calcification, characteristics that can beassessed using non-invasive imaging techniques. Subjects who are at riskof having CVD are also at risk of having an increased atheroscleoroticplaque burden, a characteristic that can be examined using intravascularultrasound.

Screening tests are of particular importance for patients with riskfactors for ischemic heart disease (IHD). A common initial screeningtest for IHD is to measure the electrical activity over a period of timewhich is reproduced as a repeating wave pattern, commonly referred to asan electrocardiograph (ECG or EKG), showing the rhythmic depolarizationand repolarization of the heart muscles. Analysis of the various wavesand normal vectors of depolarization and repolarization yields importantdiagnostic information. However, ECG measurements are not particularlysensitive nor are the data very useful for detecting cardiovascularabnormalities or malfunctions. Therefore, stressing the heart undercontrolled conditions and measuring changes in the ECG data is usually,but not always, the next step. A stress test, sometimes called atreadmill test or exercise test, can show if there's a lack of bloodsupply through the arteries that go to the heart. In a stress test, thepatient exercises under controlled conditions while various parametersare monitored, including pulse, EKG, blood pressure and tiredness. Thestresses may be applied by the performance of physical exercise oralternatively, by administration of pharmaceutical compounds such asdobutamine, which mimic the physiological effects of exercise. Anothertype of stress test used in screening tests for IED include theradionucleotide (nuclear) stress test which involves injecting aradioactive isotope (typically thallium or cardiolyte) into a patient'sbloodstream, then visualizing the spreading of the radionucleotidethroughout the vascular system and its absorption into the heartmusculature. The patient then undergoes a period of physical exerciseafter which, the imaging is repeated to visualize changes indistribution of the radionucleotide throughout the vascular system andthe heart. Stress echocardiography involves ultrasound visualization ofthe heart before, during and after physical exercise. Theradionucleotide stress test and stress echocardiography are often usedin combination with ECG measurements in order to gain a clearerunderstanding of the state of individual's cardiovascular health.

In one embodiment, elevated levels of a marker, e.g., cTnI or VEGF,detected by the devices of the present invention and the presence of aphysiological marker are indicative of a biological state, e.g., adisease. For example, a condition in a subject may be detected byelevated levels of the first marker and an irregular EKG or stress testresult.

In one embodiment, the present invention provides a method to detect ormonitor a condition in a subject, comprising detecting a first marker ina first sample from the subject and detecting a second marker, whereinthe first marker comprises Cardiac Troponin-I (cTnI) or VascularEndothelial Growth Factor (VEGF) and the second marker comprises amolecular marker. A molecular marker comprises any substance whosepresence is indicative of a biological state. Examples of molecularmarkers native to an organism include total cholesterol, high-densitylipoproteins (HDL), low-density lipoproteins (LDL) LDL/HDL ratio,triglycerides, uric acid, or creatinine. In some embodiments, themolecular marker include total cholesterol, high-density lipoproteins(HDL), low-density lipoproteins (LDL) LDL/HDL ratio, triglycerides, uricacid, or creatinine. In some embodiments, the molecular marker comprisessubfractions of LDL/HDL/Q-LDL, triglycerides. The American HeartAssociation offers the following recommendations for lipid profilemeasures:

HDL: “normal” readings vary between 40-50 mg/dL for men and 50-60 mg/dLfor women; measurements above 60 mg/dL are considered “protective.”

LDL: less than 130 mg/dL considered good; less than 100 considered“optimal”

Triglycerides: less than 150 mg/dL considered “normal”

Total Cholesterol (add ⅕ triglyceride measure to LDL and HDL numbers):under 200 mg/dL considered “desirable”

An HDL/LDL ratio between 0.3 and 0.4 or higher is generally seen asdesirable.

A molecular marker can also be introduced into a subject, e.g., rubidiumchloride is used as a radioactive isotope to evaluate perfusion of heartmuscle. Other molecular markers include blood sugar, e.g., bloodglucose, and vitamin-D.

In one embodiment, elevated levels of a marker, e.g., cTnI or VEGF,detected by the devices of the present invention and the presence of amolecular marker are indicative of a biological state, e.g., a disease.For example, a condition in a subject may be detected by elevated levelsof the first marker and a low HDL/LDL reading.

In one embodiment, the present invention provides a method to detect ormonitor a condition in a subject, comprising detecting a first marker ina first sample from the subject and detecting a second marker, whereinthe first marker comprises Cardiac Troponin-I (cTnI) or VascularEndothelial Growth Factor (VEGF) and the second marker comprises agenetic marker. A genetic marker comprises a segment of DNA with anidentifiable physical location on a chromosome whose inheritance can befollowed. Genetic markers include restriction fragment lengthpolymorphism (RFLP), amplified fragment length polymorphism (AFLP),random amplification of polymorphic DNA (RAPD), variable number tandemrepeat (VNTR), microsatellite polymorphism, minisatellites, singlenucleotide polymorphisms (SNPs), short tandem repeat (STR), and singlefeature polymorphism (SFP). Many genetic markers, e.g., SNPs, have beenlinked as risk factors for a variety of diseases. For example, one ofthe genes associated with Alzheimer's disease, apolipoprotein E (ApoE)contains two SNPs that result in three possible alleles for this gene:E2, E3, and E4. Each allele differs by one DNA base, and the proteinproduct of each gene differs by one amino acid. A person who inherits atleast one E4 allele has a greater chance of developing Alzheimer'sdisease, whereas inheriting the E2 allele seems to indicate a reducedlikelihood of developing Alzheimer's. A database of SNPs is maintainedby the HapMap project, available at http://www.hapmap.org/. Examples ofSNPs associated with cardiovascular conditions are disclosed in U.S.patent application Ser. Nos. 12/109,137; 12/139,139; 12/151,275;12/077,935; and 12/019,651. Genetic markers further comprise mutationsincluding insertions, deletions or fusions. Genetic markers furthercomprise epigenetic markers, such as DNA methylation, e.g., themethylation of a cytosine in the context of a CpG sequence. DNAmethylation patterns can be altered in cells in response to certainconditions. For example, aberrant DNA methylation is a hallmark ofcancer. Imprinting, which comprises the allele specific expression of agene, e.g., by DNA methylation silencing of one allele, can also beindicative of a condition, e.g., increased risk of a condition such ascancer. Such markers are well understood by those of skill in the art.See, e.g., Laird, Cancer epigenetics, Hum Mol Genet. 2005 Apr. 15; 14Spec No 1:R65-76; Tang and Ho, Epigenetic reprogramming and imprintingin origins of disease. Rev Endocr Metab Disord. 2007 June; 8(2):173-82.

In one embodiment, elevated levels of a marker, e.g., cTnI or VEGF,detected by the devices of the present invention and the presence of agenetic marker are indicative of a biological state, e.g., a disease.For example, a condition in a subject may be detected by elevated levelsof a first marker and a SNP correlative of the condition. For example, acondition in a subject may be detected by elevated levels of a firstmarker and a DNA methylation pattern found to correlate with thecondition.

D. Detection and Monitoring

The methods of the present invention can quantify minute changes inlevel of a biomarker, e.g., VEGF, over time when longitudinal samplesare collected from an individual over a defined period of time. Theability to quantify discreet changes is enabled by the combinedsensitivity and precision of measurements made when using the describedmethod.

The methods described herein can be used to monitor levels ofbiomarkers, e.g., VEGF, cytokines, cTnI, in healthy individuals, withthe ability to detect minute elevations in level of analyte indicativeof disease risk or early disease. Such elevations above normal can bequantified over time when regular longitudinal samples are collectedfrom an individual. The ability to monitor discreet changes is enabledby the combined sensitivity and precision of measurements made whenusing the described method.

The method described can be used to monitor levels of biomarkers, e.g.,VEGF, cytokines, cTnI, in individuals for who elevated levels have beenobserved, with the ability to detect minute decreases in the level ofanalyte indicative of a return towards a healthy state. Such decreasescan be quantified over time when regular longitudinal samples arecollected from an individual, and compared to the healthy range. Thisinformation can be used to determine success of a therapeuticintervention or a return to a normal, healthy state. The ability tomonitor discreet changes is enabled by the combined sensitivity andprecision of measurements according to the present invention.

The method described can be used to monitor minute changes in level ofanalyte, e.g., VEGF, cytokines, cTnI, over time when longitudinalsamples are collected from an individual over a defined period of time.The ability to monitor discreet changes is enabled by the combinedsensitivity and precision of measurements according to the presentinvention.

In one embodiment, the present invention provides a method to detect ormonitor a condition in a subject, comprising detecting a first marker ina first sample from the subject and detecting a second marker, whereinthe concentration of the first marker is determined and theconcentration of the second marker is determined, further comprisingmeasuring a change in concentration of the markers between the firstsample and a second sample from the subject. In some embodiments, thefirst marker comprises Cardiac Troponin-I (cTnI) or Vascular EndothelialGrowth Factor (VEGF). According to the method, the change is used todetect or monitor the condition.

In one embodiment, the present invention provides a method to detect ormonitor a condition in a subject, comprising detecting a first marker ina first sample from the subject and detecting a second marker, whereinthe concentration of the first marker is determined and theconcentration of the second marker is determined, further comprisingdetermining a change in the ratio of the concentrations of the firstmarker and the second marker between the first sample and a secondsample from the subject, whereby the change is used to detect or monitorthe condition. In some embodiments, the first marker comprises CardiacTroponin-I (cTnI) or Vascular Endothelial Growth Factor (VEGF).

In some embodiments, a medical procedure is performed between acquiringthe first sample and the second sample from the subject. In someembodiments, the medical procedure comprises a surgical procedure,stress testing, radionucleotide stress testing or a therapeuticintervention. In some embodiments, the present invention provides amethod to detect or monitor a condition in a subject, comprisingdetecting a first marker in a first sample from the subject anddetecting a second marker, performing a surgical procedure, anddetecting the first and second markers after the procedure, wherein thechange in the markers before and after the procedure is used to detector monitor the condition. In some embodiments, the first markercomprises Cardiac Troponin-I (cTnI) or Vascular Endothelial GrowthFactor (VEGF). In some embodiments, the present invention provides amethod to detect or monitor a condition in a subject, comprisingdetecting a first marker in a first sample from the subject anddetecting a second marker, performing a stress test on the subject, anddetecting the first and second markers after the stress test, whereinthe change in the markers before and after the procedure is used todetect or monitor the condition. In some embodiments, the first markercomprises Cardiac Troponin-I (cTnI) or Vascular Endothelial GrowthFactor (VEGF). In some embodiments, the present invention provides amethod to detect or monitor a condition in a subject, comprisingdetecting a first marker in a first sample from the subject anddetecting a second marker, wherein the first marker comprises CardiacTroponin-I (cTnI), performing a stress test on the subject, anddetecting the first and second markers after the stress test, whereinthe change in the markers before and after the procedure is used todetect or monitor the condition. In some embodiments, the presentinvention provides a method to detect or monitor a condition in asubject, comprising detecting a first marker in a first sample from thesubject and detecting a second marker, wherein the first markercomprises Vascular Endothelial Growth Factor (VEGF), performing a stresstest on the subject, and detecting the first and second markers afterthe stress test, wherein the change in the markers before and after theprocedure is used to detect or monitor the condition. In someembodiments, the present invention provides a method to detect ormonitor a condition in a subject, comprising detecting a first marker ina first sample from the subject and detecting a second marker,performing a therapeutic intervention on the subject, and detecting thefirst and second markers after the stress test, wherein the change inthe markers before and after the procedure is used to detect or monitorthe condition. In some embodiments, the first marker comprises CardiacTroponin-I (cTnI) or Vascular Endothelial Growth Factor (VEGF).

In one embodiment, the present invention provides a method to monitor acondition in a subject, comprising detecting a first marker in a firstsample from the subject and detecting a second marker, wherein themonitoring comprises monitoring of a disease progression, diseaserecurrence, risk assessment, therapeutic efficacy or surgical efficacy.In some embodiments, the first marker comprises Cardiac Troponin-I(cTnI) or Vascular Endothelial Growth Factor (VEGF). In someembodiments, monitoring comprises detecting the markers in a series ofsamples, e.g., two or more samples, from a subject. In some embodiments,the series of samples are collected over time at various time intervalsas disclosed herein. In some embodiments, the present inventioncomprises comparing the level of a marker from each sample from theseries of samples to the level of the marker in the sample taken fromthe first sample. In some embodiments, the series of samples arecollected from different bodily fluids, tissues, or other biologicalorigins. Such samples can be collected at identical or similar timepoints, and/or over time as above. A change in the markers or lackthereof in the series of samples can be used to monitor a biologicalstate, e.g., a disease progression, therapeutic efficacy, diseaserecurrence, risk assessment or surgical efficacy. In some embodiments,the methods comprise an analysis selected from the group consisting ofcomparing the concentration or series of concentrations of a marker ormarkers to a normal value for the concentration of the marker ormarkers, comparing the concentration or series of concentrations to abaseline value, and determining a rate of change of concentration forthe series of concentrations. In some embodiments, the methods comprisecomparing the concentration of a marker in a sample with a predeterminedthreshold concentration, and determining a diagnosis, prognosis, ormethod of treatment if the sample concentration is greater than thethreshold level.

In one embodiment, the present invention provides a method to monitor acondition in a subject, comprising detecting a first marker in a firstsample from the subject and detecting a second marker, wherein themonitoring comprises monitoring of a disease progression. In someembodiments, the first marker comprises Cardiac Troponin-I (cTnI) orVascular Endothelial Growth Factor (VEGF). In one embodiment, anincrease in a marker indicates a disease progression. In one embodiment,a decrease in a marker indicates a disease progression. In oneembodiment, lack of change in a marker indicates a disease progression.For example, an increase in a marker may indicate the growth of cellsthat express the marker, e.g., increase in a marker could indicate agrowth of tumor cells. In some embodiments, medical testing or treatmentis altered in response to the monitoring of the marker or markers. Insome embodiments, additional testing may be prescribed for the subject.For example, the results of an assay according to the present inventionmay indicate progression of cardiovascular disease and a stress test orsimilar may be ordered in response. In another example, the results ofan assay according to the present invention may indicate progression ofa cancer and an imaging technique or similar may be ordered in response.In some embodiments, a therapeutic agent or surgerical procedure may beadministered to the subject if the assay indicates disease progression.One of skill in the art will appreciate that such medical testing ortreatment will depend on the marker, condition, subject history, etc.

In one embodiment, the present invention provides a method to monitor acondition in a subject, comprising detecting a first marker in a firstsample from the subject and detecting a second marker, wherein themonitoring comprises monitoring of a disease recurrence. In someembodiments, the first marker comprises Cardiac Troponin-I (cTnI) orVascular Endothelial Growth Factor (VEGF). In one embodiment, anincrease in a marker indicates a disease recurrence. In one embodiment,a decrease in a marker indicates a disease recurrence. In oneembodiment, lack of change in a marker indicates a disease recurrence.For example, an increase in a marker may indicate the presence of cellsthat express the marker, e.g., tumor cells, thereby indicatingrecurrence of a condition, e.g., cancer. In some embodiments, medicaltesting or treatment is proscribed in response to the monitoring of themarker or markers. For example, a therapeutic agent or surgicalprocedure can be administered or performed if the assay indicatesdisease recurrence. One of skill in the art will appreciate that suchmedical testing or treatment will depend on the marker, condition,subject history, etc.

In one embodiment, the present invention provides a method to monitor acondition in a subject, comprising detecting a first marker in a firstsample from the subject and detecting a second marker, wherein themonitoring comprises monitoring of risk assessment. In some embodiments,the first marker comprises Cardiac Troponin-I (cTnI) or VascularEndothelial Growth Factor (VEGF). In one embodiment, an increase in amarker indicates a disease recurrence. In one embodiment, a decrease ina marker indicates a disease recurrence. In one embodiment, lack ofchange in a marker indicates a disease recurrence. For example, anincrease in a marker may indicate risk of, increased risk of, ordecreased risk of a cardiovascular complication, e.g., a heart attack.In some embodiments, medical testing or treatment is prescribed inresponse to the monitoring of the marker or markers. For example, atherapeutic agent or surgical procedure can be administered to thesubject if the assay indicates risk or increased risk. Likewise,therapeutic treatment may be decreased if risk has declined, e.g., inresponse to patient lifestyle changes or therapeutic efficacy. One ofskill in the art will appreciate that such medical testing or treatmentwill depend on the marker, condition, subject history, etc.

In one embodiment, the present invention provides a method to monitor acondition in a subject, comprising detecting a first marker in a firstsample from the subject and detecting a second marker, wherein themonitoring comprises monitoring of a therapeutic efficacy. In someembodiments, the first marker comprises Cardiac Troponin-I (cTnI) orVascular Endothelial Growth Factor (VEGF). In one embodiment, anincrease in a marker indicates suboptimal therapeutic efficacy. In oneembodiment, a decrease in a marker indicates suboptimal therapeuticefficacy. In one embodiment, lack of change in a marker indicatessuboptimal therapeutic efficacy. For example, an increase or lack ofchange in a marker can indicate that the therapy has failed to slow adisease progression, e.g., by being ineffective in halting tumor growth.In some embodiments, the invention provides a method of monitoring theeffectiveness of a therapeutic treatment in an individual comprisingmeasuring the concentration of a marker in a first sample from theindividual wherein the first sample is taken prior to administration ofthe therapeutic treatment and further comprising measuring theconcentration of the marker in a series of samples taken from theindividual at different time points subsequent to beginning thetherapeutic treatment and further comparing the concentration of themarker prior to the therapeutic treatment to the level of the markersubsequent to the therapeutic treatment to determine the effectivenessof the therapeutic treatment. As disclosed herein, additional markerscan be assessed to provide confirmatory or complementary results. Insome embodiments, therapeutic treatment is altered in response to themonitoring of the marker or markers. In some embodiments, the dosage ofa therapeutic agent, e.g., a drug or biological agent, may be altered inresponse to the results. In some embodiments, treatment with atherapeutic agent, e.g., a drug or biological agent, may be halted inresponse to the results. In some embodiments, additional therapeuticagents, e.g., a drug or biological agent, may be administered inaddition to or in place of the first agent in response to the results.

In one embodiment, the present invention provides a method to monitor acondition in a subject, comprising detecting a first marker in a firstsample from the subject and detecting a second marker, wherein the firstmarker comprises Cardiac Troponin-I (cTnI) or Vascular EndothelialGrowth Factor (VEGF), wherein the monitoring comprises monitoring of asurgical efficacy. In one embodiment, an increase in a marker indicatessuboptimal surgical efficacy. In one embodiment, a decrease in a markerindicates suboptimal surgical efficacy. In one embodiment, lack ofchange in a marker indicates suboptimal surgical efficacy. For example,an increase or lack of change in a marker can indicate that the surgeryfailed to remove all diseased tissue, e.g., tissue derived from a tumor.In some embodiments, the treatment of the subject is affected by theresults of the test. For example, if the results of the assay indicatethat surgical resection was unsuccessful in removing all cancer from asubject, the subject may be treated with chemotherapy. Likewise, if theresults of the assay indicate that surgical resection was successful,additional treatment may be avoided. One of skill in the art willappreciate that such medical testing or treatment will depend on themarker, condition, subject history, etc.

E. Cardiovascular Biomarker Panels

Numerous risk factors for cardiovascular disease have been identified.These risk factors can be grouped into two broad categories:unmodifiable factors (such as male gender, and family history ofpremature heart diseases) and modifiable factors. Modifiable factors canbe further subdivided into life style factors and underlyingdisorder/disease factors. In many instances lifestyle and underlyingdisease are intertwined, such as in obesity and diabetes. Combined, allof these factors can be used to identify those in the general populationwho are at especially high risk of developing cardiovascular disease.

Attempts to prevent cardiovascular disease are more effective when theyremove and prevent causes, and they often take the form of modifyingrisk factors. A non-exhaustive list of risk factors that can bemodified, either through life style changes or medicine, include thefollowing:

Life style risk factors. Cigarette smoking, obesity, and physicalinactivity.

Disease risk factors. High blood pressure, high cholesterol levels,metabolic syndrome, diabetes, rheumatoid arthritis, systemic lupuserythematosus (SLE), and obstructive sleep apnea.

High Blood Pressure. High blood pressure is a powerful risk factor forcerebrovascular disease as well as for coronary heart disease. Anestimated 50 million people have high blood pressure.

Blood Cholesterol Levels. A clear and positive relationship betweenblood cholesterol levels and subsequent coronary heart disease hasrepeatedly been demonstrated. The cholesterol level associated with thelow-density lipoprotein (LDL) fraction is positively correlated withcoronary heart disease, whereas the cholesterol associated with thehigh-density lipoprotein (HDL) is negatively correlated (the higher thelevel, the lower the risk).

Metabolic Syndrome. The main features of metabolic syndrome includeinsulin resistance, hypertension (high blood pressure), cholesterolabnormalities, and an increased risk for clotting. Patients are mostoften overweight or obese. This clustering, or development of severalrisk factors at once, leads to a greatly increased risk for CVD.

Diabetes. Diabetes is a powerful and independent risk factor forcardiovascular disease, the major cause of death in diabetic persons.Factors in addition to blood-glucose level elevate the risk.

Rheumatoid Arthritis (RA). Rheumatoid arthritis patients have a higherrisk of early death, most likely due to cardiovascular disease.

Systemic lupus erythematosus (SLE). Women with the autoimmune diseasesystemic lupus erythematosus (SLE) have a more than two-fold increasedrisk of cardiovascular disease over women without the disease.

Obstructive Sleep Apnea (OSA). OSA is characterized by repetitiveinterruption of ventilation during sleep caused by collapse of thepharyngeal airway. Population-based epidemiology studies andobservations of OSA patients have consistently shown the prevalence ofhypertension, type II diabetes, cardiovascular disease, and stroke to behigher in people with OSA.

Cardiovascular disorders, e.g., congestive heart failure (CHF), areoften first diagnosed after the onset of clinical symptoms, eliminatingpotential for early intervention. In one embodiment, the presentinvention provides a multi-marker immunoassay for more sensitive assayfor early detection of CHF in blood samples, e.g., plasma. Use of apanel of multiple markers, where the markers are quantified usingultra-sensitive single molecule counting methods, demonstrates improveddiagnostic sensitivity and specificity and can be used to improvetreatments and modification of risk factors.

In one embodiment, the present invention provides a method to assess theintegrity of heart muscle function using biomarkers. For example, theconcentration of natriuretic peptides (BNP and NTproBNP) can be used totest for and diagnose congestive heart failure in both asymptomatic andsymptomatic patients, with left ventricular dysfunction. Higherconcentrations of the peptides are associated with worse CVD outcomes;thus providing strong prognostic information. The cardiac Troponins areanother class of biomarkers that can assess cardiac muscle pathology.Troponins are produced by cardiac myocytes. Injury to these cells ineither a reversible or non-reversible manner results in the release oftroponins into circulating blood. Elevated blood troponin concentrationsin asymptomatic populations are prognostic of subsequent cardiac events,with a higher troponin concentration providing a higher riskclassification. These inflammatory cytokine blood biomarkers and heartmuscle specific blood biomarkers a means to assess CVD risk in patientsprior to CVD events as well as risk in recurring CVD. Furthermore, thesemarkers are dynamic with higher concentrations being assisted with worseoutcomes. In some embodiments, a panel of cardiac pathology and vascularinflammation biomarkers is used.

The biomarkers proBNP, cTnI, IL-6, TNF-alpha, IL-17a, and hsCRP presentat various stages of elevation in patients with various stages ofcardiovascular disease. In some embodiments, these blood biomarkers arecombined in a multi-marker panel to provide a more sensitive andspecific assessment of cardiovascular disease risk and status. In someembodiments, additional biomarkers are added to the panel. In someembodiments, two or more of the markers are used to assess CVD.Furthermore, these markers are dynamic with higher concentrations beingcorrelated with worse outcomes. These highly sensitive multi-markerpanels, wherein in the markers are quantified using single moleculedetection devices and methods as provided herein, can be combined withtraditional tests based upon physiological biomarkers for heart diseaseto assess the degree of cardiovascular risk as well as monitor at-riskpatients for improvements in disease status. Traditional diagnosticmodality testing includes, but is not limited to, treadmill/EKG stresstesting, obstructive sleep disorder (OSD) assessment, and carotid arteryintima-media thickness (CIMT) evaluation. These traditional tests areenhanced through the addition of highly sensitive blood biomarkers tocreate ancillary diagnostic modalities (ADMs), as described below.

Enhanced Stress Test. In some embodiments, the performance of a standardtreadmill stress test with EKG is enhanced with the addition of plasmacardiac troponin-I (cTnI) biomarker measurements. cTnI measuredaccording to the present invention provides insight into heart musclephysiology during cardiac stress. Plasma cTnI concentrations increase inindividuals with stress test induced ischemia, which can be measuredusing nuclear perfusion imaging and other techniques. Even mild levelsof ischemia can induce increases in cTnI concentrations. The magnitudeof increase in cTnI correlates positively with the degree of ischemia.High grade ischemia induces the highest release of cTnI from heartmuscle myocytes into blood. Using multivariable analysis comparing cTnIincrease with all other stress EKG output variables, only cTnI increasescorrelated with ischemia. Other methods to detect cTnI, e.g., Siemensultrasensitive cTnI and Roche cTnT, do not provide the sensitivity andaccuracy to correlate troponin changes with ischemia.

In some embodiments, plasma cTnI measurements are used to monitorcardio-pathology. Plasma cTnI can be measured specifically andaccurately in healthy humans, with a range of 1-8 pg/ml. Within a singleindividual, cTnI is a stable biomarker and displays minimal variation onan hourly or weekly basis. A greater than 2-fold increase in cTnIconcentration over time is significant. Moreover, a single elevatedcardiac troponin measurement is correlated with adverse cardiac events,3 months to 8 years after the measurement. Thus, the monitoring of bloodcTnI as provided herein provides a method to monitor cardiomyocyteintegrity and hence cardio-pathology, shedding insight into thelikelihood of future cardiovascular events.

Enhanced Sleep Test. Obstructive sleep disorder (OSD) is highlyprevalent yet underappreciated as a strong CVD risk factor. The AmericanHeart Association recently issued an Expert Consensus Document,highlighting the relevance of OSD to individuals who are either at riskfor or already have established CVD. See Somers V K, et al. (2008) Sleepapnea and cardiovascular disease: an American Heart Association/AmericanCollege of Cardiology Foundation Scientific Statement from the AmericanHeart Association Council for High Blood Pressure Research ProfessionalEducation Committee, Council on Clinical Cardiology, Stroke Council, andCouncil on Cardiovascular Nursing. J Am Coll Cardiol. 52:686-717. Thehallmarks of OSD include hypoxia, vasoconstriction significantlyelevated blood pressure. Such perturbations can result in vascularinflammation with increases in plasma biomarker cytokines such as TNF-a,IL-6 and IL-17a and hsCRP. These biomarker elevations can diminish withsuccessful intervention.

OSD can be diagnosed through a simple home testing procedure. Thepresent invention provides a method to enhance the home sleep test withthe addition of one or more biomarkers, e.g., plasma cytokines such asTNF-a or IL-17a. These measurements provide a broader and deeper pictureof the magnitude of sleep disorder and its impact on systemicinflammation. Because these inflammatory biomarkers are dynamic, theycan be monitored in OSD patients as a marker for successful therapeuticintervention towards both sleep apnea and vascular inflammation.

Enhanced CIMT test. Carotid intima-media thickness (CIMT) is a measureof atherosclerosis in all arteries, including the coronary artery. CIMTprovides a picture of the arterial wall, allowing assessment of thedegree of stenosis even in the case of sub-clinical atherosclerosis. Thepresent invention provides an improved CIMT test based uponultrasonography. Atherosclerosis is an end product of vascularinflammation. In some embodiments, measurement of biomarkers in blood,e.g., pro-inflammatory cytokines in plasma, can enhance CIMT byproviding physical and biochemical assessments of arterial disease riskand status, including subclinical disease. Plasma biomarkers, e.g.,hsCRP, IL-6, TNF-a and IL-1, can be elevated with vascular inflammationand correlate with stenosis measured with CIMT testing. These biomarkersprovide insight into the magnitude of inflammatory risk. Furthermore,since they are dynamic, changes in their plasma concentrations can beused to assess therapeutic effectiveness during patient monitoring.

F. Clinical Methods

The present invention relates to systems and methods (including clinicalmethods) for establishing markers that can be used for diagnosing abiological state or a condition in an organism, preparing diagnosticsbased on such markers, and commercializing/marketing diagnostics andservices utilizing such diagnostics.

In one embodiment, the clinical methods herein comprise: establishingone or more markers using a method comprising: establishing a range ofconcentrations for said marker or markers in biological samples obtainedfrom a first population by measuring the concentrations of the marker ormarkers in the biological samples by detecting single molecules of themarker or markers; and commercializing the one or more markersidentified in the above step, e.g., in a diagnostic product. Thebiomarkers identified are preferably polypeptides or small molecules.Such polypeptides can be previously known or unknown. The diagnosticproduct herein can include one or more antibodies that specificallybinds to the marker (e.g., polypeptide).

In one embodiment, the clinical methods herein comprise: establishingone or more markers using a system comprising: establishing a range ofconcentrations for said marker in biological samples obtained from afirst population by measuring the concentrations of the marker thebiological samples by detecting single molecules of the marker; andproviding a diagnostic service to determine if an organism has or doesnot have a biological state or condition of interest. A diagnosticservice herein may be provided by a CLIA approved laboratory that islicensed under the business or the business itself. The diagnosticservices herein can be provided directly to a health care provider, ahealth care insurer, or a patient. Thus the clinical methods herein canmake revenue from selling, e.g., diagnostic services or diagnosticproducts.

The clinical methods herein also contemplate providing diagnosticservices to, for example, health care providers, insurers, patients,etc. The business herein can provide diagnostic services by eithercontracting out with a service lab or setting up a service lab (underClinical Laboratory Improvement Amendment (CLIA) or other regulatoryapproval). Such service lab can then carry out the methods disclosedherein to identify if a particular marker or pattern of markers iswithin a sample.

The one or more markers are polypeptides or small molecules, or newchemical entities.

In other embodiments, data collected using the methods of the presentinvention is acquired and submitted to a medical practitioner to directa medical treatment. In an exemplary embodiment, a sample from a subjectis sent to a laboratory, wherein the sample is assayed using the methodsof the present invention. The results of the assays are thencommunicated to a medical professional, e.g., a doctor. The medicalprofessional might then direct a course of treatment for the subjectbased on the assay results. In one embodiment, the assay provides forelevated levels of cTnI or VEGF in a sample from the subject. The assayresults are submitted to the medical professional, e.g., by electroniccommunications or by standard paper mail. The medical professional cansuggest a course of therapy for the patient, e.g., a drug preventitiveof heart disease. The medical professional may also combine the assayresults with other medical markers, e.g., medical history, smoking, age,weight, race, stress testing, blood pressure, etc., when deciding acourse of action.

In some embodiments, computer systems are used to perform a variety oflogic operations of the present invention. The computer systems caninclude one or more computers, databases, memory systems, and systemoutputs (e.g., a computer screen or printer). In some embodiments,computer executable logic or program code is stored in a storage medium,loaded into and/or executed by a computer, or transmitted over sometransmission medium, such as over electrical wiring or cabling, throughfiber optics, or via electromagnetic radiation, e.g., wirelessly. Whenimplemented on a general-purpose microprocessor, the computer executablelogic can configure the microprocessor to create specific logiccircuits. In some embodiments, multiple computer systems are used. Inone embodiment, a patient or organization can provide assay data eitherby uploading such data on a secure server (meeting industry requirementsfor security) or by sending the information in a high-density portableform (such as CDROM, DVD). The data can then be analyzed at a remotelocation.

In some embodiments, the computer system comprises a computer readablemedium, e.g., floppy diskettes, CD-ROMs, hard drives, flash memory,tape, or other digital storage medium, with a program code comprisingone or more sets of instructions for performing a variety of logicoperations. In some embodiments, a computer system is used to direct theoperations of the analyzer device. In some embodiments, a computersystem is used to analyze the assay data. In some embodiments, acomputer system is used to combine the data from multiple markersthereby assising in the detection or monitoring of a biological state,e.g., a disease.

In some embodiments, a database of relevant information, e.g.,experimental protocols, marker properties or algorithms to combinemultiple markers, can be stored on a digital storage medium, e.g.,floppy diskettes, CD-ROMs, hard drives, flash memory, tape, or otherdigital storage medium. Such databases can be stored locally or remotelywith respect to other computer systems, e.g., those used to performlogic operations or present data to a medical practitioner. See FIG. 5.

VII. KITS

The invention further provides kits. In some embodiments, kits includean analyzer system and a label, as previously described. Kits of theinvention include one or more compositions useful for the sensitivedetection of a molecule, such as a marker, as described herein, insuitable packaging. In some embodiments, kits of the invention provide alabel, as described herein, together with other components such asinstructions, reagents, or other components. In some embodiments, thekit provides the label as separate components, in separate containers,such as an antibody and a fluorescent moiety, for attachment before useby the consumer. In some embodiments kits of the invention providebinding partner pairs, e.g., antibody pairs, that are specific for amolecule, e.g., a marker, where at least one of the binding partners isa label for the marker, as described herein. In some embodiments, thebinding partners, e.g., antibodies, are provided in separate containers.In some embodiments, the binding partners, e.g., antibodies, areprovided in the same container. In some embodiments, one of the bindingpartners, e.g., antibody, is immobilized on a solid support, e.g., amicrotiter plate or a paramagnetic bead. In some of these embodiments,the other binding partner, e.g., antibody, is labeled with a fluorescentmoiety as described herein.

Binding partners, e.g., antibodies, solid supports, and fluorescentlabels for components of the kits may be any suitable such components asdescribed herein.

The kits may additionally include reagents useful in the methods of theinvention, e.g., buffers and other reagents used in binding reactions,washes, buffers or other reagents for preconditioning the instrument onwhich assays will be run, filters for filtering reagents, and elutionbuffers or other reagents for running samples through the instrument.

Kits may include one or more standards, e.g., standards for use in theassays of the invention, such as standards of highly purified, e.g.,recombinant, protein markers, or various fragments, complexes, and thelike, thereof. Kits may further include instructions.

VIII. EXAMPLES

The following examples are offered by way of illustration and not by wayof limiting the remaining disclosure.

Unless otherwise specified, processing samples in the Examples wereanalyzed in a single molecule detector (SMD) as described herein, withthe following parameters: Laser: continuous wave gallium arsenite diodelaser of wavelength 639 nm (Blue Sky Research, Milpitas, Calif.),focused to a spot size of approximately 2 microns (interrogation spaceof 0.004 pL as defined herein); flow rate=5 microliter/min through afused silica capillary of 100 micron square ID and 300 micron square OD;non-confocal arrangement of lenses (see, e.g., FIG. 1A); focusing lensof 0.8 numerical aperture (Olympus); silicon avalanche photodiodedetector (Perkin Elmer, Waltham, Mass.).

Example 1 Sandwich Assays for Biomarkers: Cardiac Troponin I (cTnI)

The assay: The purpose of this assay was to detect the presence ofcardiac Troponin I (cTNI) in human serum. The assay format was atwo-step sandwich immunoassay based on a mouse monoclonal captureantibody and a goat polyconal detection antibody. Ten microliters ofsample were required. The working range of the assay is 0-900 pg/ml witha typical analytical limit of detection of 1-3 pg/ml. The assay requiredabout four hours of bench time to complete.

Materials: the following materials were used in the procedure describedbelow: Assay plate: Nunc Maxisorp, product 464718, 384 well, clear,passively coated with monoclonal antibody, BiosPacific A34440228P Lot#A0316 (5 μg/ml in 0.05 M sodium carbonate pH 9.6, overnight at roomtemperature); blocked with 5% sucrose, 1% BSA in PBS, and stored at 4°C. For the standard curve, Human cardiac Troponin I (BiosPacific Cat#J34000352) was used. The diluent for the standard concentrations washuman serum that was immonodepleted of endogenous cTNI, aliquoted andstored at −20° C. Dilution of the standards was done in a 96 well,conical, polypropylene, (Nunc product #249944). The following buffersand solutions were used: (a) assay buffer: BBS with 1% BSA and 0.1%TritonX-100; (b) passive blocking solution in assay buffer containing 2mg/ml mouse IgG, (Equitech Bio); 2 mg/ml goat IgG, (Equitech Bio); and 2mg/ml MAK33 poly, Roche#11939661; (c) detection Antibody (Ab): GoatPolyclonal antibody affinity purified to Peptide 3, (BiosPacific G129C),which was label with a fluorescent dye Alexa Fluor 647, and stored at 4°C.; detection antibody diluent: 50% assay buffer, 50% passive blockingsolution; wash buffer: borate buffer saline Triton Buffer (BBST) (1.0 Mborate, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3); elutionbuffer: BBS with 4M urea, 0.02% Triton X-100 and 0.001% BSA.

Preparation of Alexa Fluor 647 labeled antibodies: the detectionantibody G-129-C was conjugated to Alexa Fluor 647 by first dissolving100 μg of G-129-C in 400 μl of the coupling buffer (0.1M NaHCO₃). Theantibody solution was then concentrated to 50 μl by transferring thesolution into YM-30 filter and subjecting the solution and filter tocentrifugation. The YM-30 filter and antibody was then washed threetimes by adding 400 μl of the coupling buffer. The antibody wasrecovered by adding 50 μl to the filter, inverting the filter, andcentrifuging for 1 minute at 5,000×g. The resulting antibody solutionwas 1-2 μg/μl. Alexa Fluor 647 NHS ester was reconstituted by adding 20μl DMSO to one vial of Alexa Fluor 647, this solution was stored at −20°C. for up to one month. 3 μl of Alexa Fluor 647 stock solution was addedto the antibody solution, which was then mixed and incubated in the darkfor one hour. After the one hour, 7.5 μl 1M tris was added to theantibody Alexa Fluor 647 solution and mixed. The solution wasultrafiltered with YM-30 to remove low molecular weight components. Thevolume of the retentate, which contained the antibody conjugated toAlexa Fluor 647, was adjusted to 200-400 μl by adding PBS. 3 μl 10% NaN₃was added to the solution, the resulting solution was transferred to anUltrafree 0.22 centrifugal unit and spun for 2 minutes at 12,000×g. Thefiltrate containing the conjugated antibody was collected and used inthe assays.

Procedure: cTnI standard and sample preparation and analysis:

The standard curve was prepared as follows: working standards wereprepared (0-900 pg/ml) by serial dilutions of the stock of cTnI intostandard diluent or to achieve a range of cTnI concentrations of between1.2 pg/ml-4.3 μg/ml.

passive blocking solution and 10 μl of standard or of sample were addedto each well. Standards were run in quadruplicate. The plate was sealedwith Axyseal sealing film, centrifuged for 1 min at 3000 RPM, andincubated for 2 hours at 25° C. with shaking. The plate was washed fivetimes, and centrifuged until rotor reached 3000 RPM in an invertedposition over a paper towel. A 1 nM working dilution of detectionantibody was prepared, and 20 μl detection antibody were added to eachwell. The plate was sealed and centrifuged, and the assay incubated for1 hour at 25° C. with shaking. Thirty μl of elution buffer was added perwell, the plate was sealed and the assay incubated for ½ hour at 25° C.The plate was either stored for up to 48 hours at 4° C. prior toanalysis, or the sample was analyzed immediately.

For analysis, 20 μl per well were acquired at 40 μl/minute, and 5 μlwere analyzed at 5 μl/minute. The data were analyzed based on athreshold of 4 sigma. Raw signal versus concentration of the standardswas plotted. A linear fit was performed for the low concentration range,and a non-linear fit was performed for the full standard curve. Thelimit of detection (LoD) was calculated as LOD=(3× standard deviation ofzeros)/slope of linear fit. The concentrations of the samples weredetermined from the equation (linear or non-linear) appropriate for thesample signal.

An aliquot was pumped into the analyzer. Individually-labeled antibodieswere measured during capillary flow by setting the interrogation volumesuch that the emission of only 1 fluorescent label was detected in adefined space following laser excitation. With each signal representinga digital event, this configuration enables extremely high analyticalsensitivities. Total fluorescent signal is determined as a sum of theindividual digital events. Each molecule counted is a positive datapoint with hundreds to thousands of DMC events/sample. The limit ofdetection the cTnI assay of the invention was determined by the mean+3SD method.

Results: Data for a typical cTnI standard curve measured inquadruplicate using the assay protocol is shown in Table 3.

TABLE 3 Standard Curve for cTnI cTnI Standard % (pg/ml) Signal DeviationCV 0 233 25 10.8 1.5625 346 31 8.9 3.125 463 35 7.5 6.25 695 39 5.6 12.51137 61 5.3 25 1988 139 7.0 50 3654 174 4.8 100 5493 350 6.4 200 8264267 3.2 400 9702 149 1.5 800 9976 50 0.5

The sensitivity of the analyzer system was tested in 15 runs and wasfound routinely to detect sub femtomolar (fM) levels of calibrator, asshown by the data in Table 4. The precision was 10% at 4 and 12 pg/mlcTnI.

TABLE 4 Instrument Sensitivity Calibrator Signal (fM) counts CV 0 11 12302 9 60 1341 8 300 4784 7

Linearized standard curve for the range concentrations of cTnI are shownin FIG. 6.

The analytical limit of detection (LoD) was determined across 15sequential assays. The LoD was the mean of the 0 std+3 SD (n=4)intra-assay determinations. The average LoD was 1.7 pg/ml (range 0.4-2.8pg/ml).

The recovery of the sample was determined by analyzing samples of serumthat had been immunodepleted of cTnI and spiked with known amounts ofcTnI. Table 5 shows the data for sample recovery by the system analyzedover 3 days.

TABLE 5 Sample Recovery Spike Recovery Standard % (pg/ml) (mean)Deviation CV 5 5.7 0.9 16 15 13.7 0.2 2 45 43 0.6 2 135 151 6.2 4

The linearity of the assay was determined in pooled human serum that wasspiked with cTnI and diluted with standard diluent. The results in Table6 show the dilutions and % of the signal expected for the correspondingdilution.

TABLE 6 Assay Linearity Serum Dilution % of expected 1:2 79 1:4 87 1:896

In further experiments, the present invention provides cTnIquantification to normal levels, e.g., 0.8 pg/ml at a CV of 10% andless. The analytical sensitivity of the assay system for cTnI ispresented graphically in FIG. 7A. The LoD was between 0.1-0.2 pg/ml. For100 μl samples, the LoD was 0.117 pg/ml. For a 50 μl sample the LoD was0.232 pg/ml. The low end standard curve signal is shown in FIG. 7B.

These data show that the analyzer system of the invention allows forperforming highly sensitive laser-induced immunoassay for sub-femtomolarconcentrations of cTnI. The assay can be used to equilaterally quantifycTnI across humans, rats, dogs and monkeys.

Example 2 Sandwich Bead-Based Assays for TnI

The assays described above use the same microtiter plate format wherethe plastic surface is used to immobilize target molecules. The singleparticle analyzer system also is compatible with assays done in solutionusing microparticles or beads to achieve separation of bound fromunbound entities.

Materials: MyOne Streptavidin C1 microparticles (MPs) are obtained fromDynal (650.01-03, 10 mg/ml stock). Buffers use in the assay include: 10×borate buffer saline Triton Buffer (BBST) (1.0 M borate, 15.0M sodiumchloride, 10% Triton X-100, pH 8.3); assay buffer (2 mg/ml normal goatIgG, 2 mg/ml normal mouse IgG, and 0.2 mg/ml MAB-33-IgG-Polymer in 0.1MTris (pH 8.1), 0.025 M EDTA, 0.15 M NaCl, 0.1% BSA, 0.1% Triton X-100,and 0.1% NaN₃, stored at 4° C.); and elution buffer (BBS with 4M urea,0.02% Triton X-100, and 0.001% BSA, stored at 2-8C). Antibodies used inthe sandwich bead-based assay include: Bio-Ab (A34650228P (BiosPacific)with 1-2 biotins per IgG) and Det-Ab (G-129-C (BiosPacific) conjugatedto A647, 2-4 fluors per IgG). The standard is recombinant human cardiactroponin I (BiosPacific, cat #J34120352). The calibrator diluent is 30mg/ml BSA in TBS wEDTA.

Microparticles Coating: 100 μl of the MPs stock is placed in aneppendorf tube. The MPs are washed three times with 100 μl of BBST washbuffer by applying a magnet, removing the supernatant, removing themagnet, and resuspending in wash buffer. After the washes the MPs areresuspended in 100 μl of assay buffer and 15 μg of Bio-Ab are added. Themixture is then incubated for an hour at room temperature with constantmixing. The MPs are washed five times with 1 ml wash buffer as describedabove. After the washes the MPs are resuspended in 15 ml of assay buffer(or 100 μl to store at 4° C.).

Preparation of Standard and Samples: The standard is diluted withcalibrator diluent to prepare proper standard curve (usually 200 pg/mldown to 0.1 pg/ml). Frozen serum and plasma samples need to becentrifuged 10 minutes at room temperature at 13K rpm. Clarifiedserum/plasma is removed carefully to avoid taking any possible pelletsor floaters and put into fresh tubes. 50 μl of each standard or sampleis pippetted into appropriate wells.

Capture Target: 150 μl of MPs (after resuspension to 15 ml in assaybuffer +400 mM NaCl) are added to each well. The mixture is incubated onJitterBug, 5 at room temperature for 1 hr.

Washes and Detection: The plate is placed on a magnet and thesupernatant is removed after ensuring that all MPs are captured by themagnet. 250 μl of wash buffer are added after removing the plate fromthe magnet. The plate is then placed on the magnet and the supernatantis removed after ensuring that all MPs are captured by the magnet. 20 μlDet-Ab are added per well (Det-Ab to 500 ng/ml is diluted in assaybuffer +400 mM NaCl)). The mixture is incubated on JitterBug, 5 at roomtemperature for 30 min.

Washes and Elution: The plate is placed on a magnet and washed threetimes with wash buffer. The supernatant is removed after ensuring thatall MPs are captured by the magnet and 250 μl of wash buffer are added.After the washes the samples are transferred into a new 96-well plate.The new plate is then placed on the magnet and the supernatant isremoved after ensuring that all MPs are captured by the magnet. 250 μlof wash buffer are then added after removing the plate from the magnet.The plate is then placed on the magnet and the supernatant is removedafter ensuring that all MPs are captured by the magnet. 20 μl of elutionbuffer are then added and the mixture is incubated on JitterBug, 5 atroom temperature for 30 min.

Filter out MPs and transfer to 384-well plate: The standard and samplesare transferred into a 384-well filter plate placed on top of a 384-wellassay plate. The plate is then centrifuged at room temperature at 3000rpm with a plate rotor. The filter plate is removed and the appropriatecalibrators are added. The plate is covered and is ready to be run onSMD.

SMD: An aliquot is pumped into the analyzer. Individually-labeledantibodies are measured during capillary flow by setting theinterrogation volume such that the emission of only 1 fluorescentmolecule is detected in a defined space following laser excitation. Witheach signal representing a digital event, this configuration enablesextremely high analytical sensitivities. Total fluorescent signal isdetermined as a sum of the individual digital events. Each moleculecounted is a positive data point with hundreds to thousands of DMCevents/sample. The limit of detection the cTnI assay of the invention isdetermined by the mean+3 SD method.

Example 3 Concentration Range for cTnI in a Population of NormalNon-Diseased Subjects

A reference range or normal range for cTnI concentrations in human serumwas established using serum samples from 88 apparently healthy subjects(non-diseased). A sandwich immunoassay as described in Example 1 wasperformed and the number of signals or events as described above werecounted using the single particle analyzer system of the invention. Theconcentration of serum troponin I was determined by correlating thesignals detected by the analyzer with the standard curve as describedabove. All assays were perfumed in quadruplicate.

In accordance with recommendations by the current European and AmericanCardiology Societies (ESC/ACC) troponin assays should quantifyaccurately the 99th percentile of the normal range with an assayimprecision (CV) of less than 10% in order to distinguish reliablybetween patients with ACS and patients without ischemic heart disease,and risk stratification for adverse cardiac events. The assay showedthat the biological threshold (cutoff concentration) for TnI is at a TnIconcentration of 7 pg/ml, which is established at the 99th percentilewith a corresponding CV of 10% (FIG. 8). At the 10% CV level theprecision profile points at a TnI concentration of 4 and 12 pg/ml.

In addition, the assay correlates well with the Troponin-I standardmeasurements provided by the National Institute of Standards andTechnology (FIG. 9).

The assay of the invention is sufficiently sensitive and precise tofulfill the requirements of the ESC/ACC, and it is the most sensitiveassay for cardiac troponin I when compared to assays such as thosedescribed by Koerbin et al., Ann Clin Biochem, 42:19-23 (2005). Theassay of the invention has a 10-20 fold greater sensitivity thancurrently available assays, which has determined the biologicalthreshold range to be 111-333 pg/ml cTnI.

Example 4 Detection of Early Release of TnI into the Circulation ofPatients with Acute Myocardial Infarction (AMI)

Study 1: 47 samples were obtained serially from 18 patients thatpresented with chest pain in the emergency department (ED). Thesepatients all had non-ST elevated ECG were, and were diagnosed with AMI.The concentration of cTnI in the initial samples from all 18 patientswas determined according to a commercial assay at the time of admissionto the emergency room to be <350 pg/ml (10% cutpoint), and 12 were <100pg/ml (99th %) percentile. These samples were tested at later timesusing the same commercial assay, and were determined to test positivefor cTnI. The same serum samples were also assayed for TnI according tothe assay of the invention as described in Examples 1 and 3, and theresults compared to the results obtained using the commercial assay.

Blood was drawn for the first time at the time the patient presentedwith chest pain (sample 1), and subsequently at intervals between 4-8hours (samples 2 at 12 hours; sample 3 at 16 hours; sample 4 at 24hours; sample 5 at 30 hours; sample 6 at 36 hours; sample 7 at 42 hours;and sample 8 at 48 hours). The serum was analyzed by the methods of theinvention and by a current commercial method, and the results obtainedare shown in FIG. 10. The analyzer of the invention detected TnI at thetime the patient presented with chest pain (sample 1), while thecommercial assay first detected cTnI at a much later time (sample 6 at36 hours). The concentration of TnI in sample 3 exceeded the biologicalthreshold level that was established using the analyzer of the invention(7 pg/ml, see FIG. 8), and indicated that sample 3 is positive for TnIto suggest the incidence of a cardiac event. The biological thresholdfor the commercial assay lies between 111 and 333 pg/ml of TnI.Accordingly, sample 3 would not have been considered to indicate apossible cardiac event.

In addition, the methods and compositions of the present invention allowfor much earlier diagnosis and possible intervention based on cardiactroponin levels, as evidenced by results for the first sample taken fromthe patients. In the 3 cases that had initial commercial assay cTnIvalues of between 100 and 350 ng/ml, all were positive for cTnI by theanalytical methods of the invention (i.e., cTnI over 7 pg/ml). In the 12cases that had initial commercial cTnI values of less than 100 pg/ml, 5were determined to be positive for a cardiovascular event according tothe assay of the invention (i.e., cTnI over 7 pg/ml). The prospectiveuse of the assay of the invention would have detected 53% more AMI casesthan the current commercial assay when the admission sample was tested.

Study 2: 50 additional serum samples, which tested negative according tothe commercial assay, were tested using the analyzer and assay of theinvention. The results are shown in FIG. 11. Of the 50 samples, 36 werewithin the 99th % and determined to be within the normal rangeestablished by the assay of the invention. However, the remaining 14samples that were determined to be within the commercial “normal” ornon-diseased range, tested above the biological threshold established bythe invention.

Therefore, the high sensitivity cTnI assay of the invention allows forthe detection of myocardial damage in patients when cTnI serum levelsare below threshold values by commercially available technology. The useof the highly sensitive and precise cTnI assay of the invention enablesdetection of AMI earlier than with existing cTnI assays, and therebyprovides the opportunity for appropriate diagnosis and early medicalintervention to improve the outcome.

Example 5 Detection of Leukotriene T4 (LTE4)

The assay was developed to quantify Leukotriene E₄ (LTE₄) in buffer as apreliminary assay for assays using, e.g., urine specimens. The assayformat was a one-step single antibody competitive immunoassay. Fiftymicroliters of sample were required. The typical working range of thisassay was 0-300 pg/ml with a typical limit of detection of 2-3 pg/ml(0.1-0.15 pg/sample). The assay required about four hours of bench timeto complete.

The following materials were prepared and used in the proceduredescribed below: Mouse anti-rabbit IgG coated plate provided in CaymanChemical Leukotriene E₄ (EIA Kit, Catalog #520411); stock LTE₄ Standard(purified LTE4 at 100 ng/ml in ethanol (Cayman Chemical Leukotriene E₄EIA Kit, Catalog #520411)); assay buffer (10×EIA buffer concentrate(Cayman Chemical Leukotriene E₄ EIA Kit, Catalog #520411)) diluted 1:10with 90 ml Nanopure water; buffer for dilution of standards (3%ethanol); anti-LTE₄ antibody (Leukotriene E₄ EIA antiserum (CaymanChemical Leukotriene E₄ EIA Kit, Catalog #520411) diluted with 30 ml EIAbuffer; streptavidin-Alexa detection reagent stock solution of 31M(streptavidin labeled with Alexa Fluor™ 647); tracer (LTE₄-biotinconjugate) was made compatible for detection by the analyzer; washbuffer (400× concentrate (Cayman Chemical Leukotriene E₄ EIA Kit,Catalog #520411)) diluted 1:40; elution buffer (borate buffered saline,pH 8.3 with 4M urea, 0.02% Triton X-100 and 0.001% BSA). The matrix ofthe tracer and the antiserum concentrations were tested to identify themost sensitive assay conditions.

A standard curve was prepared as follows: working standards wereprepared by making serial dilutions of the 100 ng/ml stock into assaybuffer to achieve a range of concentrations between 0.005 pg/ml and 3000pg/ml. 50 μl standard (or sample) were added per well of the assayplate. All standards were run in duplicate. Working tracer was preparedby diluting the tracer stock to 1 pg/ml with assay buffer. 50 μl tracer(or buffer) were added per well of the assay plate. A 10% workingantiserum solution was prepared by diluting 100% stock (made accordingto the kit instructions) into assay buffer. 50 μl antiserum (or buffer)were added per well of the assay plate; the plate was sealed andincubated overnight at 25° C. with shaking. A working streptavidin-Alexadetection reagent was prepared by diluting stock to 140 pM with assaybuffer. 15 μl of detection reagent were added to each well, and theplate was incubated for 30 min at 25° C. with shaking. The plate waswashed 5 times. 50 μl of elution buffer were added to each well, and theplate was incubated for ½ hour at 25° C. with shaking. The plate was useimmediately or stored for up to 48 hours at 4° C. prior to analysis.

20 μl were pumped into the analyzer at a rate of 40 μl/minute, and 5 μlof sample were analyzed at 5 μl/minute. The data files were analyzedusing a threshold=4 sigma, and a cross correlation of between 0-8 msec.Raw signal versus concentration was plotted for the standards, and alinear fit was used for low range standards, while a non-linear fit wasused for full standard curve. The limit of detection was calculated asLOD=80% of the maximum signal (no target control) (the concentration atwhich B/B₀=80%). The concentrations of samples were calculated from theequation (linear or non-linear) appropriate for the sample signal.

The competition curve of LTE4 is shown in FIG. 12. The LOD wascalculated to be 80% B/Bo=1.5 pg/ml (approximately 5 pM). The LTE4 assayperformed using a commercially available kit can detect LTE4 only ifpresent at a concentration of at least 30 pg/ml.

Therefore, the analyzer system can be used to detect levels of LTE4 toindicate the presence of an LTE4-related disorder, e.g., asthma at theonset of disease, and alert clinicians to the need for therapeuticintervention at an early stage of the disease to improve the clinicaloutcome.

Example 6 Detection of Human Akt1

A sandwich immunoassay was developed for the quantification of lowlevels of Akt1 in serum samples. A standard curve was generated bydilution of a concentrated standard into a buffered protein solution.Ten microliters (μl) of assay buffer and 10 μl of sample or standardwere added to each well of a 384-well plate that had been coated with anantibody specific for Akt1 and incubated for two hours. Morespecifically antibody 841660 (R&D Systems) was coated onto Nunc Maxisorpplates at 2.5 micrograms/ml. The plate was washed, and 20 μl of labeleddetection antibody specific for Akt1, AF1775 (R&D Systems), labeled withAlexa Fluor 647, 2-4 fluors/IgG, was added to each well. After one hourof incubation the plate was washed to remove unbound detection antibody.Bound detection antibody was eluted and measured in the analyzerinstrument.

The following materials were used in the assay procedure describedbelow. Coated 384 well plate; assay buffer; resuspension buffer;dilution buffer; standard diluent; Akt1 standard; detection antibodyreagent for Akt1; wash buffer (10 mM Borate, 150 mM NaCl, 0.1%TritonX-100, pH 8.3); elution buffer (4 M urea with 0.02% Triton X-100and 0.001% BSA), Microplate shaker set at “7”, Microplate washer, Platecentrifuge, Axyseal sealing film, Axygen product 321-31-051, Nuncpierceable sealing tape, Nunc product 235306.

Materials: Provided Reagents

Capture antibody: 841660 (R&D Systems), coated onto Nunc Maxisorp plates@ 2.5 micrograms/ml (384 well plate)Assay buffer

Resuspension Buffer Dilution Buffer

Standard diluentAkt 1 standardDetection antibody reagent for Akt1, AF1775 (R&D Systems), labeled withAlexa Fluor 647, 2-4 fluors/IgG

Other Required Reagents

TritonX-100 Wash buffer (10 mM Borate, 150 mM NaCl, 0.1% TritonX-100, pH8.3)

Elution buffer (4 M urea with 0.02% Triton X-100 and 0.001% BSA)

Microplate shaker, set at “7”

Microplate washer

Plate centrifuge

Axyseal sealing film, Axygen product 321-31-051

Nunc pierceable sealing tape, Nunc product 235306

Procedure:

Akt1 standard preparation

Resuspend standard in 0.5 ml Resuspension Buffer, finalconcentration=170 ng/ml

Dilute standard 1:3 in Dilution Buffer=57 ng/ml

Dilute standard 1:19 in Standard Diluent=3 ng/ml

Do serial 3 fold dilutions down to 4.1 pg/ml in Standard DiluentAdd 10 μl Assay Buffer per wellAdd 10 μl standard or sample per wellSeal plate with Axyseal sealing film

Spin 1 min at 3000 RPM

Incubate 2 hours at 25° C. with shakingWash plate five timesSpin plate inverted on a paper towel 1 min at 3000 RPMAdd 201 detection antibody reagent per wellSeal plate with Axyseal sealing filmSpin plate inverted on a paper towel 1 min at 3000 RPMIncubate 1 hour at 25° C. with shakingWash plate five timesSpin plate inverted on a paper towel 1 min at 3000 RPMAdd 301 elution buffer per well

Spin 1 min at 3000 RPM

Seal with Nunc pierceable sealing tape, secure tight seal with rollerIncubate ½ hour at 25° C. with shakingThe plate may be stored for up to 48 hours at 4° C. prior to analysisAnalyze on ZeptX instrument

The Akt1 standard curve was generated as follows. Akt1 standards wereprepared to achieve a range of between 4.1 pg/ml to 170 ng/ml Akt1. 10μl of each standard dilution (or sample) were added to the assay platewells. The plate was sealed and incubated for 2 hours at 25° C. withshaking. The plate was washed and centrifuged dry. 20 μl detectionantibody reagent was added per well and incubated for 1 hour at 25° C.with shaking. The antibody-Akt1 complex was disrupted by adding 30 μlelution buffer per well and incubating for ½ hour at 25° C. withshaking. The plate was either used immediately or stored for up to 48hours at 4° C. prior to analysis. Eluate was pumped into the analyzer.

Data for a typical Akt1 standard curve measured in quadruplicate usingthe assay protocol is given in Table 7, and the graphed data is shown inFIG. 13.

TABLE 7 Standard curve for Akt1 Concentration Akt1 standard AverageStandard % (pg/ml) Signal deviation CV 0 113 16 14 4.1 126 10 8 12.4 1331 0 37 151 34 22 111 173 15 8 333 350 74 21 1000 733 136 19 3000 1822243 13

Intra-Assay Precision was tested using 36 replicate samples of the 1000pg/ml standard by assaying the samples on a single plate. The averagesignal was 1822±243 with a % CV=13. The limit of detection of the assay(LoD) was determined by adding two standard deviations to the meansignal of thirty six zero standard replicates and calculating thecorresponding Akt1 concentration from the standard curve. The LoD wascalculated to be 25 pg/ml.

Therefore, the analyzer system can be used to detect levels of Akt1 todetermine the presence or absence of an Akt1-related disorder, e.g.,cancer.

Example 7 Detection of TGF-β

A sandwich immunoassay was developed for the quantification of lowlevels of TGFβ in serum. A standard curve was generated by dilution of aconcentrated standard into a buffered protein solution. Ten microliters(μl) of assay buffer and 10 μl of sample or standard were added to eachwell of a 384-well plate coated with an antibody specific for TGFβ andincubated for two hours. The plate was washed and 20 μl of labeleddetection antibody specific for TGFβ was added to each well. After 1 hof incubation the plate was washed to remove unbound detection antibody.Bound detection antibody was eluted and measured in the analyzerinstrument.

The following materials were used in the assay procedure describedbelow. Coated 384 well plate; assay buffer; standard diluent; 10 μg/mlstock solution of TGFβ standard; detection antibody reagent for TGFβ;TritonX-100 Wash buffer (100 mM Borate, 150 mM NaCl, 0.1% TritonX-100,pH 8.3); elution buffer (4M urea with 0.02% Triton X-100 and 0.001%BSA).

The TGF-β standard curve was generated as follows. TGF-β standards wereprepared to achieve a range of between 100 ng/ml to 4.1 pg/ml TGFβ. 10μl assay buffer and 10 μl standard or sample were added to each well.The plate was sealed and incubated for 2 hours at 25° C. with shaking.The plate was sealed and incubated for 2 hours at 25° C. with shaking.The plate was washed and centrifuged dry. 20 μl detection antibodyreagent was added per well and incubated for 1 hour at 25° C. withshaking. The antibody—TGF-β complex was disrupted by adding 30 μlelution buffer per well and incubating for ½ hour at 25° C. withshaking. The plate was either used immediately or stored for up to 48hours at 4° C. prior to analysis. Eluate was pumped into the analyzer.

Data for a typical TGF-β standard curve measured in quadruplicate usingthe assay protocol is given in Table 8, and the graphed data is shown inFIG. 14.

TABLE 8 Standard curve for TGF-β Concentration Average Standard %(pg/ml) Signal deviation CV 0 1230 114 9 4 1190 68 6 12 1261 132 10 371170 158 14 111 1242 103 8 333 1364 135 10 1000 1939 100 5 3000 3604 49714

The limit of detection of the assay (LoD) was determined by adding twostandard deviations to the mean signal of twenty zero standardreplicates and calculating the corresponding TGFβ concentration from thestandard curve. The LoD=350 pg/ml.

Therefore, the analyzer system can be used to detect levels of TGFβ todetermine the presence or absence of a TGFβ-related disorder, e.g.,cancer.

Example 8 Detection of Fas Ligand

A sandwich immunoassay for the quantification of low levels of Fasligand in serum. A standard curve was generated by dilution of aconcentrated standard into a buffered protein solution. Ten microliters(μl) of assay buffer and 10 μl of sample or standard were added to eachwell of a 384-well plate coated with an antibody specific for Fas ligandand incubated for 2 hours. The plate was washed and 20 μl of labeleddetection antibody specific for Fas ligand was added to each well. After1 hour incubation the plate was washed to remove unbound detectionantibody. Bound detection antibody was eluted and measured in the ZeptX™instrument.

The Fas ligand standard curve was generated as follows. Fas ligandstandards were prepared to achieve a range of between 100 ng/ml to 4.1pg/ml Fas ligand. 10 μl assay buffer and 10 μl standard or sample wereadded to each well. The plate was sealed and incubated for 2 hours at25° C. with shaking. The plate was sealed and incubated for 2 hours at25° C. with shaking. The plate was washed and centrifuged dry. 20 μldetection antibody reagent was added per well and incubated for 1 hourat 25° C. with shaking. The antibody-Fas ligand complex was disrupted byadding 30 μl elution buffer per well and incubating for ½ hour at 25° C.with shaking. The plate was either used immediately or stored for up to48 hours at 4° C. prior to analysis.

Data for a typical Fas ligand standard curve measured in quadruplicateusing the assay protocol is given in Table 9.

TABLE 9 Standard curve for Fas ligand Concentration Fas Average Standard% ligand standard (pg/ml) Signal deviation CV 0 935 82 9 1.2 1007 44 43.4 1222 56 5 11 1587 70 4 33 2869 52 2 100 5939 141 2 300 9276 165 2900 11086 75 1

Intra-Assay Precision was tested using 12 replicate samples of 3standard concentrations by assaying the samples on a single plate. Themean, standard deviation and CV for the 12 values for each of the threepoints are shown in Table 10.

TABLE 10 Intra-assay precision for Fas ligand Concentration AverageStandard % (pg/ml) Signal deviation CV 11 1717 128 7 33 3031 262 9 1006025 257 4

The limit of detection of the assay (LoD) was determined by adding twostandard deviations to the mean signal of twenty zero standardreplicates and calculating the corresponding Fas ligand concentrationfrom the standard curve. The LoD was calculated to be 2.4 pg/ml.

Therefore, the analyzer system of the invention can detect levels of Fasligand to indicate the presence of a Fas ligand-related disorder, e.g.,cancer, allograft rejection and degenerative diseases such asosteoarthritis.

Example 9 Sandwich Assays for Biomarker TREM-1

Assays for TREM-1 have been developed using a sandwich assay format(Sandwich Assay for Detection of Individual Molecules, U.S. ProvisionalPatent Application No. 60/624,785). Assay reagents for TREM-1 detectionare available commercially (R&D Systems, Minneapolis, Minn.). The assaywas done in a 96 well plate. A monoclonal antibody was used as thecapture reagent, and either another monoclonal or a polyclonal antibodywas used for detection. The detection antibody was labeled with AlexaFluor 647®.

The assay protocol was as follows:

1. Coat plates with the capture antibody, washed 5×,

2. Block in 1% BSA, 5% sucrose in PBS,

3. Add the target diluted in serum, incubate, wash 5×,

4. Add the detection antibody, incubate, wash 5×

5. Add 0.1M glycine pH 2.8 to release the bound assay components fromthe plate.

6. Transfer samples from the processing plate to the detection plate,bring the pH of the sample to neutral and run on the single particleanalyzer system.

FIG. 16 shows a standard curve of TREM-1 generated using the assay. Theassay was linear in the measured range of 100-1500 femtomolar. An ELISAassay from R&D Systems has recently been introduced. The standard curvereported for their ELISA assay is between 60-4000 pg/ml. This Examplesuggests we can routinely measure 100 fM (4.7 pg/ml) in a standardcurve, allowing for about 100× more sensitive measurements. In addition,standard curves for chemokines, T cell activation molecules, celladhesion molecules and signal transduction molecules have beengenerated. See FIG. 18. The results show that the detection by thedetection of analyte using the single particle analyzer is consistentlybetween 10- and 100-fold more sensitive than detection using ELISAassays.

Example 10 Sandwich Assays for Biomarkers: IL-6 and IL-8 Levels in Serum

The assay: This protocol describes a sandwich immunoassay for thequantification of low levels of IL-6 in serum using the single particleanalyzer system of the invention. A standard curve was generated bydilution of a concentrated standard into a buffered protein solution.Ten microliters (μl) of assay buffer and 10 μl of sample or standardwere added to each well of a 384-well plate coated with an antibodyspecific for IL-6 and incubated for two hours. The plate was washed, and20 μl of labeled detection antibody specific for IL-6 was added to eachwell. After one hour of incubation the plate was washed to removeunbound detection antibody. Bound detection antibody was eluted andmeasured in the single particle analyzer instrument.

Materials: The following materials were used in the procedure describedbelow: coated 384 well plate; assay buffer; standard diluent; 100 ng/mlstock solution of IL-6 standard; detection antibody for IL-6 (R&DSystems) labeled with Alexa Fluor 647 dye; TritonX-100 Wash buffer (10mM Borate, 150 mM NaCl, 0.1% TritonX-100, pH 8.3); Elution buffer (4 Murea with 0.02% Triton X-100 and 0.001% BSA); Microplate shaker set at“7”; Microplate washer; Plate centrifuge; Axyseal sealing film, Axygenproduct 321-31-051; and Nunc pierceable sealing tape, Nunc product235306.

Procedure: A standard curve for IL-6 was prepared as follows: 100 ng/mlstock solution was thawed and diluted 1:1000 to 100 pg/ml in standarddiluent by doing six serial, 3 fold dilutions to obtain a range ofconcentration having the lowest standard concentration of 0.14 pg/ml. 10μl assay buffer and 10 μl standard or sample were added to each well perwell of the coated 384 well plate. The plate was sealed with Axysealsealing film, and centrifuged for one minute at 3000 RPM. The assayplate was incubated for 2 hours at 25° C. with shaking; washed fivetimes; and centrifuged while inverted on a paper towel for one minute at3000 RPM. 20 μl detection antibody reagent was added to each well; theplate was sealed with Axyseal sealing film, and centrifuged for oneminute at 3000 RPM. The assay plate was incubated for one hour at 25° C.with shaking, washed five times, and centrifuged while inverted on apaper towel for one minute at 3000 RPM. 30 μl elution buffer was addedto each well; the plate was sealed with Nunc pierceable sealing tape,and a tight seal was secured using with roller. The assay plate wascentrifuged for one minute at 3000 RPM, and incubated for ½ hour at 25°C. with shaking. Analysis of the assay was performed immediately.Alternatively, the plate was stored for up to 48 hours at 4° C. prior toanalysis.

Samples of serum from EDTA treated whole blood of 32 blood bank donorswere analyzed for IL-6.

Results: Data for a typical IL-6 standard curve measured inquadruplicate using the assay protocol is shown in Table 11.

TABLE 11 Standard Curve for IL-6 Concentration Average Standard (pg/ml)Signal deviation CV 370 11035 206 2% 125 9983 207 2% 41 8522 95 1% 145023 108 2% 4.5 2577 124 5% 1.7 1178 114 10%  0.5 577 36 6% 0 106 1514% 

Linearized standard curves for higher and low range concentrations ofIL-6 are shown in FIGS. 17A-B, respectively. The assay allowed fordetection of IL-6 at less than 0.5 pg/ml (FIGS. 17A-B). The limit ofdetection (LoD) was calculated to be 0.06 pg/ml. The limit of detectionof the assay (LoD) was determined by adding two standard deviations tothe mean signal of the zero standard replicates and calculating thecorresponding IL-6 concentration from the standard curve. This level ofsensitivity is excellent for detection of even normal levels of IL-6which ranges between 0.5 and 10 pg/ml.

Assays to detect IL-6 and IL-8 in serum of blood samples from blood bankdonors were performed, and the results of the analysis are shown inFIGS. 17C-D. IL-6 was quantified in 100% of the samples (32/32). Theaverage concentration of IL-6 was 2.3 pg/ml, and the range ofconcentration was 0.2 to >26 pg/ml (FIG. 17C). The same samples werealso assayed for IL-8 essentially using the procedure described forIL-6. IL-8 standards and IL-8 specific antibodies were used. A standardcurve for IL-8 was established (not shown) and used to determine theconcentration of IL-8 in the samples (FIG. 17D). IL-8 was quantified in100% (32/32) samples. The average concentration for IL-8 was 7.3 pg/ml,and the range of concentration was 1.2 to >26 pg/ml.

Measurements of IL-6 or any particle of interest can be measured at lowand higher concentrations (FIGS. 17A and B) by switching the detectionof the analyzer from counting molecules (digital signal) to detectingthe sum of photons (analog signal) that are generated at the higherconcentrations of analyte. This is shown in a general way in FIG. 17E.The single particle analyzer has an expanded linear dynamic range of 6logs. The ability to increase the dynamic range for detecting theconcentration of a particle in a sample allows for the determination ofthe concentration of a particle for normal (lower concentration range)and disease levels (higher concentration range). The range of detectionfor normal and disease levels of IL-6 is shown in FIG. 17F.

Example 11 Vascular Endothelial Growth Factor-A (VEGF-A) Assay

Assays to detect VEGF were developed for both human VEGF and mouse VEGF.In some embodiments, the human VEGF assay has an LOD of about 0.1 pg/mland an LLOQ of 0.3 pg/ml, making it 90× more sensitive than the commonlyused ELISA assay. Cross-reactivity with mouse VEGF was minimal for allsample types tested. The assay was capable of measuring VEGFconcentrations in 100% of the plasma, cell lysate, and spent mediasamples tested. In contrast, an ELISA was typically able to accuratelydetect human VEGF in only 6% of healthy plasma samples, and 10% ofhealthy cell lysate samples. Where both assays measured the VEGFconcentration in a sample, the levels determined were comparable for thetwo assays, with the exception of spent media where the ELISA detectedconsiderably more VEGF. This discrepancy is likely due to the fact thatthe ELISA measures total VEGF while the assay of the present inventionmeasures free VEGF. Soluble VEGF receptors released into the spent mediawould significantly decrease the free VEGF concentration. Theintra-assay variability was <10% for most plasma samples, and <15% forplasma samples with high VEGF concentrations. Inter-assay CVs foranalysis of plasma samples was <10%.

Example 12 Sandwich Immunoassay for the Detection of Mouse and HumanVEGF Preparation of Antibody and Antigen Reagents

Generation the necessary antibody and antigen reagents required fordeveloping the mouse VEGF bioassays. To identify optimal reagents forthe mouse VEGF assay, recombinant mouse VEGF protein (from R&D Systemsand Sigma) and anti-mouse VEGF antibodies (from R&D Systems, Abcam, andSigma) were tested for suitability. For the human VEGF assay recombinantVEGF protein (from R&D Systems and Abcam) and anti-human VEGF antibodies(from R&D Systems and Abcam) were obtained and tested. Magneticparticles were coated with anti-VEGF antibodies for use in the capturestep of the sandwich-immunoassay format. Potential detection antibodieswere conjugated with Alexa Fluor® dye. Antibody pairs for both assayswere screened as part of the assay optimization process using a basicset of initial assay conditions.

Preparation of Sandwich VEGF Immunoassay

Using optimal antibody pairs as prepared in the preparation of antibodyand antigen reagents, assays were run to optimize the concentrations forcapture antibody, detection antibody, and magnetic particles. Inaddition, various assay components were tested to design the optimalassay buffers for each assay. This included identifying the bestblocking agents and detergents, then optimizing the concentrations ofeach component.

Methods for Performing Human VEGF Assay:

A solution of recombinant human VEGF protein standard at a concentrationof 1 ng/ml was serially-diluted. Triplicate samples were prepared. TheVEGF assay was used to measure the concentrations of these samples. Theconcentrations determined using the assay were plotted against theexpected VEGF concentration.

Results:

The performance of the assays were demonstrated and found to providehighly linear correlation with the concentration of input recombinantVEGF used as standards. The human VEGF assay has an LOD of 0.1 pg/ml andan LLOQ of 0.3 pg/ml (Table 12 and FIGS. 19A-B). Table 12 shows humanVEGF assay performance data wherein the assay demonstrates a CVs<10%,and recoveries of 84-107%.

TABLE 12 Detected hVEGF Events std (pg/ml) (Mean) dev CV Recovery 0.24197 8 4%  95% 0.48 311 11 3% 100% 0.98 484 27 6%  89% 1.95 885 40 5% 93% 3.9 1537 57 4%  90% 7.8 2975 225 8% 116% 15.6 4972 110 2% 114% 31.37349 70 1% 111% 62.5 9401 95 1% 114% 125 10023 96 1% 100% 250 10091 1602%  84% 500 10236 99 1%  95% 1000 10029 34 <1%  107%

Table 13 shows human VEGF assay performance data wherein the assaydemonstrates an LOD of 0.07 pg/ml.

TABLE 13 BACKGROUND SLOPE (DETECTED [(DETECTED LOD EVENTS)EVENT)/(PG/ML)] [PG/ML] 81 202 0.07

The data presented in Tables 12 and 13 are shown graphically in FIGS.19A-B.

Similarly, the mouse VEGF assay has an LOD of 2 pg/ml and an LLOQ of 3pg/ml (Tables 14 and 15).

TABLE 14 mVEGF Observed % % [pg/ml] mVEGF stdev CV recovery 1000 982 10611 98 250 256 12 5 102 63 62 4 7 99 16 15 3 19 94 3.9 7.7 3 36 197

TABLE 15 StDev LoD 10% LoD Slope Bkg bkg [pg/ml] [pg/ml] 12 217 30 3.64.9

The data presented in Tables 14 and 15 are shown graphically in FIGS.20A-B.

The data demonstrate that the mouse VEGF assay is 3× more sensitive andthe human VEGF assay is 90× more sensitive when compared to the statedsensitivities of the respective benchmark R&D Systems VEGF ELISA assaykits (mVEGF assay sensitivity of 9 pg/ml; human VEGF assay sensitivityof 32 pg/ml). [Note that the R&D Systems stated LOD of 6.8 pg/ml forhuman VEGF assay must be multiplied by 5 to accurately define the LOD ofthe assay. This adjustment is needed to account for the 1:5 dilution ofthe samples required in the R&D Systems assay (the standards in thisassay are not diluted, and the 1:5 dilution of the sample is notincluded as part of their sensitivity calculation)]. For the human VEGFassay of the Example, magnetic particles coated with a monoclonalantibody are used for the capture step and an Alexa-labeled polyclonalantibody is used for the detection step. For the mouse VEGF assaypolyclonal antibodies are used for both the capture and detection steps,similar to the R&D Systems ELISA kit.

In order to ensure equal comparison between the present invention andthe ELISA assay, a comparison was made between standard analyteconcentrations used for value assignment. As a result of thisinformation, the standard according to the present invention wasrevalued in accordance with results from the assay of the ELISAStandards. When the present data were adjusted for thisstandard-revaluation, the VEGF concentrations determined using bothassays were similar. The original and re-valued data are presented inTable 16 below.

Example 13 Determination of Reproducibility, Variability, and Accuracyof Human and Mouse VEGF Biomarker Assays in Plasma Comparison ofAnalysis of Human Plasma:

Plasma samples from 24 individual mice were analyzed using an assayaccording to the present invention; 12 of these samples also were testedusing the R&D Systems ELISA (claimed sensitivity of LoD=31.2 pg/mL inserum/plasma). The assay of the invention determined the VEGFconcentration of all 12 samples, whereas the ELISA assay quantified only1/12 (8.3%) of the tested samples (Table 16 and FIG. 21). Table 16 showsthe comparison between the assay and ELISA human VEGF assays for plasmaanalysis.

TABLE 16

ND = none detected NT = not tested Shaded = tested in both assays

Select date shown in Table 16 is illustrated graphically in FIG. 21 as acomparison between Singulex and ELISA assays of human plasma. The ELISAassay detected VEGF in one sample (1 of 16 tested); VEGF values for theother plasma samples were below the lowest point on the ELISA standardcurve and therefore could not be reliably determined. CVs for Singulexassay averaged <20%.

Determination of hVEGF Levels in Cell Lysates and Culture Media inDifferent Cell Lines:

Two different human cell lines were grown and harvested. Cells werelysed according to the NCI SOP #340506 with the exception that a lowerconcentration of SDS was used and the samples were not boiled. The celllines used were human cell lines MDA-MB-231 breast adenocarcinoma andHT-29 colon adenocarcinoma.

Samples were run in duplicate in both the present and the R&D ELISAassays. Lysates were initially diluted 1:8, then (3) serial 1:2dilutions were made. Media were analyzed neat and diluted 1:4, 1:16, and1:64. Duplicates of each sample were tested. A comparison of the valuesfrom the two assays is shown in FIG. 21. Both assays detected VEGF inthe cell extracts and in the spent media (FIGS. 22A-B). Assay resultswere in general agreement, with less VEGF detected in the cell extractsthan in the spent media. Overall VEGF levels were significantly lower inthe MDA-MD231 samples, and this was confirmed by both assays.

Comparison of Analysis of Mouse Plasma Samples:

Eight mouse plasma samples from individual mice were analyzed using anassay of the present invention and the R&D Systems ELISA. Comparablevalues were observed in both of the assays (FIG. 23).

Determining mVEGF Levels in Cell Lysates and Culture Media:

Three different mouse cell lines were grown and harvested. Cells werelysed as above. The cell lines used were mouse cell lines: B16 melanoma,4T1 mammary carcinoma, and CT26 colon carcinoma.

Samples were run in duplicate in both the present and the R&D ELISAassays. Lysates were initially diluted 1:8, then (3) serial 1:2dilutions were made. Media were analyzed neat and diluted 1:4, 1:16, and1:64. Duplicates of each sample were tested. A comparison of the valuesfrom the two assays is shown in FIG. 24. Both assays detected VEGF inthe cell extracts and in the spent media. Assay results were in similarranges for each of the cell lines and are shown in FIGS. 24A-C. As seenbetween the figures, 4T1 mammary cell line had the lowest levels; B16melanoma samples had about 4× the levels of 4T1, and CT26 colon sampleswere about twice as high as for B16. There was a consistent differencebetween the two assays. The present assay detected more VEGF in the celllysates and less in the spent media. In addition, the ratio of cellularVEGF to released VEGF was consistently about 5:1 for the Singulex assay,but the ratio of intracellular VEGF to extracellular VEGF variedconsiderably for the ELISA assay results.

Example 14 VEGF Intra-Assay and Inter-Assay Performance

Human VEGF (hVEGF) Intra-Assay Reproducibility:

Sample Preparation:

Two different normal human plasma samples were assayed multiple timesusing a single microassay plate. The P1 plasma sample was assayed neatand as a 1:8 dilution. The diluted plasma provided a source of samplesto determine intra-assay at low hVEGF concentrations. Samples weretested in replicates of 18, 21, and 18.

Results:

A summary of intra-assay reproducibility for human plasma samples isshown in Table 17. The data summary in Table 17 indicates CVs for thesample replicates as 7, 12, and 9%. The last column in Table 17 showsthe corrected, measured VEGF concentrations based upon benchmarking theconcentration of the assay standards relative to the standards used inthe ELISA assay. VEGF concentrations under 2 pg/ml were measured with aCV<10%.

TABLE 17 Measured Detected hVEGF Corrected Plasma Events [pg/ml] stdhVEGF Sample (mean) std dev CV N mean dev CV [pg/ml] P1  933  53  6% 21 6 0.4  7%  1.9 diluted P1 4692 395  8% 21 41 5.0 12% 12.6 P2 4502 62614% 21 40 3.4  9% 12.3

Human VEGF Inter-Assay Reproducibility: Sample Preparation:

To test the inter-assay reproducibility of the standard curves andvalues for human plasma samples assays were independently run 7 times bydifferent personnel over 3 days with 3 replicates per sample.

Results:

The inter-assay variability between human plasma samples is shown inTable 18. Coefficients of Variation (CVs) for the plasma assays wereunder 10% (Table 18). CVs for the plasma analyses likewise were under10% with the exception of the plasma P2 results for assay Run #6 (Table18). In this assay two of the three values were in close agreement andone of the values was substantially lower. If this one replicate wereremoved from the series, the overall CV's for the VEGF plasma sampleanalyses would be <10%.

TABLE 18 INTERASSAY INTERASSAY CALCULATIONS CALCULATIONS (N = 7) Meas-Meas- sured Inter- sured Inter- MEASURED HVEGF hVEGF assay hVEGF assayPlasma [PG/ML] Barb's mean Std Sample mean Std Sample Sample Run 1 Run 2Run 3 Run 4 Run 5 Run 6 Run [pg/ml] Dev CV N [pg/ml] Dev CV N P1 26 2323 20 23 22 20 23 2  9% 6 22 2  9% 7 P2 16 16 17 18 16 10 16 16 3 19% 616 3 17% 7 P3 26 29 28 24 29 27 26 27 2  7% 6 27 2  6% 7 P4  8  9 10  9 9 10  9  9 1  7% 6  9 1  6% 7

Mouse VEGF Intra-Assay Reproducibility: Sample Preparation:

Replicate samples from three different EDTA mouse plasma were assayed ona single microtiter plate according to the present invention.

Results:

The intra-assay reproducibility for mouse plasma samples is shown inTable 19. Data for the 18-21 individual replicates from each plasmasample are shown in Table 19. CVs for the replicates of the three plasmasamples ranged from 14% to 16% (Table 19).

TABLE 19 DETECTED MEASURED MOUSE EVENTS STD MVEGF [PG/ML] STD PLASMAMEAN DEV CV MEAN DEV CV M1 3979 525 13% 485 78 16% M2 2682 349 13% 30043 14% M3 4838 516 11% 635 91 14%

Singulex Inter-Assay Reporducibility—Mouse Plasma Samples SamplePreparation:

Four different mouse EDTA plasma samples were clarified bycentrifugation for 10 minutes at 13,000×g. The samples were then testedin triplicate on 6 different days.

Results:

A summary of the inter-assay reproducibility of the mouse plasma VEGFassay is shown in Tables 20 & 21. The CVs for the mouse plasma sampleswere <25% over the six assays (Tables 20 & 21).

TABLE 20 MEASURED MVEGF MOUSE [PG/ML] STD PLASMA MEAN DEV CV M1 825.5159.2 19% M2 2107.0 422.8 20% M3 1342.4 341.2 25% M4 2582.8 398.7 15%

TABLE 21 BACK INTERPOLATED VALUES − Mouse MVEGF [PG/ML] Plasma Run 1 Run2 Run 3 Run 4 Run 5 Run 6 M1 1134  756  704  758  745  857 M2 2044 25911785 1730 1814 2677 M3 1282  908 1027 1738 1705 1394 M4 2760 2361 30442550 2853 1929

Example 15 VEGF in Xenograft Mice

Samples from mouse breast cancer xenografts were obtained from thelaboratory of Dr. Matthew Ellis at Washington University. Plasma andbreast cancer tissue was obtained from five different xenograft lines.As controls, plasma and mouse breast tissue from SCID mice were used.All samples were tested for the presence of mouse VEGF and human VEGF.Mouse VEGF ranged from 86-109 pg/ml in normal mouse plasma. Three of thexenograft mice had VEGF levels twice as high as the normals, and theother two xenograft samples had VEGF levels on the low side of theapparent normal range (80-86 pg/ml). Data are

Table 22 mVEGF Back Interpolation assay Detected Events (DE) MeasuredMouse Detected Events std mVEGF [pg/ml] std Plasma mean dev CV N meandev CV N1 1029  91  9% 3 100 14 14% N2  882 152 17% 3  92 21 22% N3 1046146 14% 3 107 17 16% N4  867 150 17% 3  86 18 21% N5 1066 116 11% 3 10914 13% T1 1733  64  4% 3 191  8  4% T2 1875 168  9% 3 210 22 10% T3 2022194 10% 3 228 25 11% T4  822  61  7% 3  80  7  9% T5  886 114 13% 3  8813 15% N = Normal mouse heparin plasma T = Mouse (xenograft) heparinplasma

Example 16 Immunoassay Kit for the Quantitative Determination of HumanVEGF in Plasma and Cellular Lysates

The Erenna™ Human VEGF Immunoassay uses a quantitative fluorescentsandwich immunoassay technique to measure Vascular Endothelial GrowthFactor (VEGF) in human plasma and cellular lysates. A capture antibodyspecific for human VEGF has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards and samples intouncoated microplate wells. During incubation, the free VEGF present inthe sample binds to the capture antibody on the coated MP. Unbound VEGFmolecules are washed away during the subsequent buffer exchange and washsteps. Fluorescent-labeled dye detection antibody specific for VEGF isadded to each well and incubated. This detection antibody will recognizeand bind to VEGF that has been captured onto the MP. During thefollowing wash step the MP's are transferred to a clean plate. Elutionbuffer is then added and incubated. The elution buffer dissociates thebound protein sandwiches from the MP surface. The fluorescent antibodiesare now free-floating in the wells. These antibodies are separated fromthe microparticles during transfer to a final microplate and the plateis loaded into the Erenna System where the fluorescent molecules arecounted. The number of fluorescently-labeled detection antibodiescounted is directly proportional to the amount of free VEGF present inthe sample when captured. The amount of free VEGF in unknown samples isinterpolated off of a standard curve.

Reagents Provided

TABLE 23 Reagent Data COMPONENT ITEM SHIPPING STORAGE PART # DESCRIPTIONCONDITIONS CONDITIONS NUMBERS 1. Human VEGF Standard Diluent With coldpack 2-8° C. 02-0182-00 2. Human VEGF Capture Reagent With cold pack2-8° C. 02-0187-00 3. Human VEGF Detection Reagent With cold pack 2-8°C. 02-0188-00 4. Erenna^(tm) VEGF Human N/A Ambient Immunoassay KitInstructions 5. Human VEGF Standard On Dry Ice ≦−70° C.  02-0180-00(frozen, shipped in separate box) 6. 10X Wash Buffer With cold pack 2-8°C. 02-0180-00 7. Elution Buffer With cold pack 2-8° C. 02-0002-02

1. Storage Instructions & Stability

The Erenna VEGF Reagent Kit is to be stored at 2-8° C. The standard isshipped on dry ice in a separate container and should be stored at ≦−70°C. It is important that the standard remain frozen upon kit arrival. Theexpiration date of the kit components can only be guaranteed if thecomponents are stored properly, and if each component is used once.Components are labeled with appropriate expiration dates.

2. Additional/Other Supplies

TABLE 24 Consumables and Supplies COMPONENT ITEM MFR PART PACKAGING ##DESCRIPTION SUPPLIER NUMBERS PRODUCT USES DETAIL 1. Erenna ™ 10X SystemsSingulex 02-0111-00, Systems (Analysis) 1 L (10 L mixed) Buffer02-0111-01 Buffer, fluid used to 2 L (20 L mixed) run Erenna System 2.Reservoirs for 12-Channel VWR 80092-466 Transfer of reagents 10/pkgPipetters 3. 96-Well V-Bottom PP Axygen P-96-450V-C or Additional assay10 plates/unit Plate, 500 μL P-96-450V-C-S plate, dilutions 5 units/case4. 96-Well Deep Well PP Axygen P-2ML-SQ-C, Prepare standard VariablePlate (2.2 mL, 1.64 mL or P-DW-20-C or curves (choose size) 1.09 mL)P-DW-11-C 5. 384-Well Round Bottom Nunc 264573 Receiver/analysis 20/pkor PP, 120 μL plate 120/cs 6. AcroPrep ™ 384-Filter Pall 5070 Remove MPsfrom 10/pkg Plates, 100 μL, for sample assay preparation and detection7. Advanced Pierceable Nunc 235306 Permanent seal for 100 units/pkSealer, Polyethylene analysis plate, used 100 pks/cs prior to Erenna run8. AxySeal-PCRSP Plate Axygen PCR-SP Sealing plates during 100 films/sealing film series incubation/mix/store case

3. Microparticle Parts and Supplies

TABLE 25 Microparticle Hardware ITEM MFR COMPONENT PKG # DESCRIPTIONSUPPLIER PART ## PRODUCT USES DETAIL 1. Dynal MPC ® - 96S Dynal ™ 120.27Rare Earth Magnet, 1 plate capture MP during wash 2. Microplate WashStation — Wash MP following — capture on magnet 3. Centrifuge w/PlateRotor — Remove MP via filter 1 plate ≧ 3000 RPM 4. Centrifuge AdapterCollar Pall 5225 Creates fit b/n 384-well 2/pkg filter plate 384-wellassay plate 5. Vacuum Pump Welch 2511B-01 Degassing systems 1 buffer 6.Microplate Incubator/ Boekel # 130000 The Incubating plate 1 ShakerScientific Jitterbug ™ 7. Plate Seal Roller, VWR VWR 60941-118 Securesplate seal 1 Plate Roller, Film + Foil permanent plate seal CS1#

4. Other Useful Supplies (Unspecified)

-   -   De-ionized or distilled water    -   Multichannel pipette capable of transferring or adding 20 μL,        100 μL and 250 μL    -   Micro-centrifuge tubes    -   Mini-centrifuge    -   250 mL container    -   250 mL graduated cylinder

Precautions

-   -   Always use caution when handling any biological samples by        wearing protective clothing and gloves.    -   Components of this reagent kit contain approximately 0.1% of        sodium azide as a preservative. Sodium azide is a toxic and        dangerous compound when combined with acids or metals. Solutions        containing sodium azide should be disposed of properly.

Technical Hints Due To High Sensitivity of Assay

-   -   Wipe down bench and pipettes with 70% Isopropanol before use.    -   Quick spin concentrated standard and initial standard dilution        before opening vials.    -   Use sterile pipette tips and reagent trays to help avoid        cross-contamination.    -   Use filter tips while transferring concentrated standard.    -   It is recommended to use a 96-well 1 mL polypropylene dilution        plate for preparing standards and samples.    -   It is recommended to transfer 3 replicates of each standard        point from the dilution plate then into the 96-well VEGF Assay        Plate.    -   Pre-wet tips (aspirate and dispense within well) twice before        each transfer.

Reagent Preparation

-   -   1. Warm all reagents to room temperature prior to use.    -   2. Prepare 1× Wash Buffer (from 10× Wash Buffer) as follows:        -   a. Pour 25 mL bottle of 10× Wash Buffer into 250 mL            container.        -   b. Add 225 mL of de-ionized water.        -   c. Mix thoroughly by gentle inversion.    -   3. Re-suspend MP by inverting the vial via a rotator for 30        minutes immediately prior to use to help ensure that the MP are        evenly distributed in the vial.

Assay Preparation Standard—Initial Standard Dilution Directions

-   -   1. Vortex and quick spin standard vial in a mini-centrifuge        prior to opening vial. Use care when opening this concentrated        standard vial to prevent loss of materials or aerosol        contamination of specimens or plates.    -   2. Refer to Certificate of Analysis for Standard for        concentration of the VEGF standard. Dilute the stock to 10 ng/mL        with Standard Diluent.

5. Plasma Sample Standard Curve

Prepare standard curve into a column on a 96-well 1 ml deep dilutionplate. Perform 1:2 serial dilutions to achieve a curve from 200 pg/ml to0.05 pg/ml. Run the standards in triplicate.

C. Cell Lysates and Media Standard Curve

Prepare standard curve into a column on a 96-well 1 ml deep dilutionplate. Perform 1:2 serial dilutions to achieve a curve from 4000 pg/mlto 0.24 pg/ml. Run the standards in triplicate.

D. Sample Preparation

It is critical that plasma samples are centrifuged at >15,800×g for 10minutes immediately prior to use. Carefully pipette, avoidingparticulates; slowly aspirate below the lipid layer. Avoid repeatedfreeze-thaw cycles. Add samples to the 96-well plate for ease intransferring.

Lysates should be centrifuged at 4,600×g for 5 minutes at 4° C.immediately prior to use. Carefully pipette the supernatant. Avoidfreeze-thaw cycles.

Lysates should be diluted at least 10 fold into standards diluent priorto loading onto the assay.

Human VEGF Assay Procedure Assay Setup

Perform the Reagent Preparation per instructions included in the kit andbulk reagent package inserts. Prepare the standard curve and samples asdescribed above.

Target Capture

After micro particles (MP) have been re-suspended, add 100 μL of VEGFCapture Reagent to 96-well polypropylene plate (PPP). Pipette 100 μL perwell of Standards/Samples to 96-well PPP. Seal plate with a temporaryplate seal (AxySeal, PCRSP Plate Sealing Film) or equivalent.Incubate/shake at medium setting for 2 hours at room temperature (RT).Carefully remove temporary plate seal to avoid splashing. Set plate ontomagnet (Dynal MPC®-96S), wait 2 minutes for MP to settle (ensure all MPare amassed as a pellet by magnet), then aspirate the supernatant (MPremain visible). With the MP secured, add 250 μL of Wash Buffer to eachwell. Wait 2 minutes (MP remain amassed) and aspirate buffer.

Detection

Remove plate from the magnet and add 20 μL of VEGF Detection Reagent toeach well. Seal plate with temporary seal. Pulse in centrifuge up to100×g. Remove the plate from the centrifuge and incubate/shake for 1hour at (RT). Remove plate seal and set plate onto magnet. Wait 2minutes and aspirate the supernatant. Add and then remove 250 μL of WashBuffer 3 times (3×) while MP are magnetized/amassed. Pause for 2 minutesafter each buffer addition. Do not suspend or remove MP from the magnet.Remove plate from the magnet and add 250 μL of Wash Buffer to each well.Shake plate for 10 seconds to re-suspend MP. Transfer contents of eachwell to a new 96-well PPP. Set a new 96-well plate onto magnet and wait2 minutes for MP to amass/settle. Remove Wash Buffer. Remove plate frommagnet, add 250 μL of Wash Buffer and shake for 10 sec. Load plate onmagnet, wait 2 minutes, then aspirate buffer. Repeat cycle, magnetizedMP should be visible.

Elution

Remove plate from the magnet and add 20 μL of per well Elution Buffer.Seal plate with temporary seal and pulse in centrifuge up to 100×g.Incubate/shake for 30 minutes at RT. Separately, set a 384-wellfilter-plate over a 384-well polypropylene plate making a filter-bottomplate using a centrifuge adapter column. Remove seal from 96-well plate,allow the MP to mass for 2 minutes while on the magnet beforetransferring the specimens to the 384-well filter-bottom plate. Coverthe top of the filter-bottom plate with temporary plate seal and setplates into centrifuge. Spin plates at 850×g for 1 minute at RT. Removefilter plate and discard, cover assay plate using the piercable(permanent) plate seal (Nunc, 235306). To ensure a good seal, use PlateSeal Roller (VWR #60941-118). Load completed assay plate onto ErennaImmunoassay System.

Human VEGF Quick Assay Guide

-   -   1. Prepare all reagents, standard curve, and samples as        instructed.    -   2. Add 100 μL of Capture Reagent, followed by 100 μL of        Standards/Samples to each well of 96-well polypropylene plate.    -   3. Cover and incubate/shake for two hours at RT.    -   4. Remove cover, set plate onto magnet, allow 2 minutes for MP        to settle/amass and remove supernatant.    -   5. With plate on magnet, add 250 μL of Wash Buffer. Wait 2        minutes and remove buffer.    -   6. Remove from magnet and add 20 μL of VEGF Detection Reagent        per well. Pulse centrifuge at 100×g.    -   7. Cover and incubate/shake for 1 hour at RT.    -   8. Set plate onto magnet and wait 2 minutes for MP to amass.        Remove supernatant.    -   9. Add and then remove 250 μL of Wash Buffer 3× with MP        magnetically amassed near the magnet. Wait 2 minutes before        aspirating the buffer between each cycle.    -   10. Remove from magnet, add 250 μL of Wash Buffer and shake        plate for 10 seconds to re-suspend MP. Transfer entire contents        to new 96-well plate.    -   11. Set plate onto magnet, wait 2 minutes. Remove supernatant.    -   12. Remove from magnet, add 250 μL of Wash Buffer and shake        plate for 10 seconds.    -   13. Repeat steps 11 and 12 respectively.    -   14. Remove from magnet and add 20 μL of Elution Buffer to each        well. Pulse centrifuge at 100×g.    -   15. Cover and incubate/shake at RT for 30 minutes.    -   16. Set a filter plate over 384-well plate (assay plate).    -   17. Transfer contents of 96-well plate to 384-well filter        plate/assay plate combo.    -   18. Cover filter plate combo, centrifuge for 1 minute at 850×g.    -   19. Remove top filter plate and discard. Cover 384-well plate        with pierceable plate seal cover.    -   20. Load the plate onto the Erenna System.

Additional Sample Information

This assay may be used to test various types of plasma and serum.

Performance Characteristics Typical Standard Curve

The Standard Curve shown in Table 26 is provided for informationalpurposes. A standard curve should be generated for each set of samplesassayed.

TABLE 26 Standard Curve Expected hVEGF [pg/ml] DE mean std dev cv EPmean std dev cv TP mean std dev cv 0.0  153  18 12%  17199  2170 13% 6615461  28222 0% 0.6  418  12  3%  41438  3697  9%  6663194  13673 0%1.2  636  8  1%  59525  2384  4%  6692487  124775 2% 2.4 1153  76  7% 112940  13057 12%  6842728  106599 2% 4.8 1885 192 10%  183411  16850 9%  6974526  119540 2% 9.7 3263 212  6%  366427  18106  5%  7541609 60299 1% 19.5 5552 112  2%  778196  35582  5%  8686751  29150 0% 39.07342 213  3% 1323456  45408  3%  10765907  264979 2% 78.3 8803 258  3%2168907  90905  4%  14280963  82371 1% 156.3 9371 170  2% 3233333  71024 2%  22666397 1186338 5% 312.5 9683 179  2% 4813765 103021  2%  373555271599907 4% 625.0 9691 268  3% 6064170  88983  1%  63385314  816036 1%1,250.0 9607  11  0% 7597939  35178  0% 107624478 4993201 5% 2,500.09203 149  2% 8444198 467406  6% 168143795 7431591 4% KEY: DetectedEvents (DE), Event Photons (EP), Total Photons (TP)

Example 17 Immunoassay Kit for the Quantitative Determination of MouseVEGF in Plasma and Cellular Lysates

The Erenna™ Mouse VEGF Immunoassay uses a quantitative fluorescentsandwich immunoassay technique to measure Vascular Endothelial GrowthFactor (VEGF) in mouse plasma and cellular lysates. A capture antibodyspecific for mouse VEGF has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards and samples intouncoated microplate wells. During incubation, the VEGF present in thesample binds to the capture antibody on the coated MP. Unbound VEGFmolecules are washed away during the subsequent buffer exchange and washsteps. Fluorescent-labeled dye detection antibody is added to each welland incubated. This detection antibody will recognize and bind to VEGFthat has been captured onto the MP. During the following wash step theMP's are transferred to a clean plate. Elution buffer is then added andincubated. The elution buffer dissociates the bound protein sandwichesfrom the MP surface. The fluorescent antibodies are now free-floating inthe wells. These antibodies are separated during transfer to a finalmicroplate and the plate is loaded into the Erenna System where thefluorescent molecules are counted. The number of fluorescently-labeleddetection antibodies counted is directly proportional to the amount ofVEGF present in the sample when captured. The amount of VEGF in unknownsamples is interpolated off of a standard curve.

Reagents Provided

TABLE 27 Reagent Data COMPONENT ITEM SHIPPING STORAGE PART # DESCRIPTIONCONDITIONS CONDITIONS NUMBERS 8. Mouse VEGF Standard Diluent With coldpack 2-8° C. 02-0207-00 9. Mouse VEGF Capture Reagent With cold pack2-8° C. 02-0201-00 10. Mouse VEGF Detection Reagent With cold pack 2-8°C. 02-0205-00 11. Erenna^(tm) VEGF Mouse N/A Ambient Immunoassay KitInstructions 12. Mouse VEGF Standard On Dry Ice ≦−70° C.  02-0200-00(frozen, shipped in separate box) 13. 10X Wash Buffer With cold pack2-8° C. 02-0179-00 14. Elution Buffer With cold pack 2-8° C. 02-0002-02

Storage Instructions & Stability

The Erenna VEGF Reagent Kit is to be stored at 2-8° C. The standard isshipped on dry ice in a separate container and should be stored at ≦−70°C. It is important that the standard remain frozen upon kit arrival. Theexpiration date of the kit components can only be guaranteed if thecomponents are stored properly, and if each component is used once.Components are labeled with appropriate expiration dates.

Additional/Other Supplies

TABLE 28 Consumables and Supplies COMPONENT ITEM MFR PART PACKAGING #DESCRIPTION SUPPLIER NUMBERS PRODUCT USES DETAIL 1. Erenna ™ 10X SystemsSingulex 02-0111-00, Systems (Analysis) 1 L (10 L mixed) Buffer02-0111-01 Buffer, fluid used to 2 L (20 L mixed) run Erenna System 2.Reservoirs for 12-Channel VWR 80092-466 Transfer of reagents 10/pkgPipetters 3. 96-Well V-Bottom PP Axygen P-96-450V-C or Additional assay10 plates/unit Plate, 500 μL P-96-450V-C-S plate, dilutions 5 units/case4. 96-Well Deep Well PP Axygen P-2ML-SQ-C, Prepare standard VariablePlate (2.2 mL, 1.64 mL or P-DW-20-C or curves (choose size) 1.09 mL)P-DW-11-C 5. 384-Well Round Bottom Nunc 264573 Receiver/analysis 20/pkor PP, 120 μL plate 120/cs 6. AcroPrep ™ 384-Filter Pall 5070 Remove MPsfrom 10/pkg Plates, 100 μL, for sample assay preparation and detection7. Advanced Pierceable Nunc 235306 Permanent seal for 100 units/pkSealer, Polyethylene analysis plate, used 100 pks/cs prior to Erenna run8. AxySeal-PCRSP Plate Axygen PCR-SP Sealing plates during 100 films/sealing film series incubation/mix/store case

Microparticle Parts and Supplies

TABLE 29 Microparticle Hardware ITEM MFR COMPONENT PKG ## DESCRIPTIONSUPPLIER PART # PRODUCT USES DETAIL 1. Dynal MPC ® - 96S Dynal ™ 120.27Rare Earth Magnet, 1 plate capture MP during wash 2. Microplate WashStation — — Wash MP following — capture on magnet 3. Centrifuge w/PlateRotor — — Remove MP via filter 1 plate ≧ 3000 RPM 4. Centrifuge AdapterCollar Pall 5225 Creates fit b/n 384-well 2/pkg filter plate 384-wellassay plate 5. Vacuum Pump Welch 2511B-01 Degassing systems 1 buffer 6.Microplate Incubator/ Boekel # 130000 The Incubating plate 1 ShakerScientific Jitterbug ™ 7. Plate Seal Roller, VWR VWR 60941-118 Securesplate seal 1 Plate Roller, Film + Foil permanent plate seal CS1#

Other Useful Supplies (Unspecified)

-   -   De-ionized or distilled water    -   Multichannel pipette capable of transferring or adding 20 μL,        100 μL and 250 μL    -   Micro-centrifuge tubes    -   Mini-centrifuge    -   250 mL container    -   250 mL graduated cylinder

Precautions:

Always use caution when handling any biological samples by wearingprotective clothing and gloves. Components of this reagent kit containapproximately 0.1% of sodium azide as a preservative. Sodium azide is atoxic and dangerous compound when combined with acids or metals.Solutions containing sodium azide should be disposed of properly.

Technical Hints Due To High Sensitivity of Assay:

Wipe down bench and pipettes with 70% Isopropanol before use.

-   -   Quick spin concentrated standard and initial standard dilution        before opening vials.    -   Use sterile pipette tips and reagent trays to help avoid        cross-contamination.    -   Use filter tips while transferring concentrated standard.    -   It is recommended to use a 96-well 1 mL polypropylene dilution        plate for preparing standards and samples.    -   It is recommended to transfer 3 replicates of each standard        point from the dilution plate then into the 96-well VEGF Assay        Plate.    -   Pre-wet tips (aspirate and dispense within well) twice before        each transfer.

Reagent Preparation

Warm all reagents to room temperature prior to use. Prepare 1× WashBuffer (from 10× Wash Buffer) as follows: Pour 25 mL bottle of 10× WashBuffer into 250 mL container; Add 225 mL of de-ionized water; Mixthoroughly by gentle inversion. Re-suspend MP by inverting the vial viaa rotator for 30 minutes prior to use to ensure that the MP are evenlydistributed in the vial.

Assay Preparation Standard—Initial Standard Dilution Directions

Vortex and quick spin standard vial in a mini-centrifuge prior toopening vial. Use care when opening this concentrated standard vial toprevent loss of materials or aerosol contamination of specimens orplates. Refer to Certificate of Analysis for Standard for concentrationof the VEGF standard. Dilute the stock to 10 ng/mL with StandardDiluent.

Standard Curve

Prepare standard curve into a column on a 96-well 1 ml deep dilutionplate. Perform 1:2 serial dilutions to achieve a curve from 4000 pg/mlto 3.9 pg/ml. Run the standards in triplicate.

Sample Preparation

Plasma samples are centrifuged at >15,800×g for 10 minutes immediatelyprior to use. Carefully pipette, avoiding particulates; slowly aspiratebelow the lipid layer. Avoid repeated freeze-thaw cycles. Add samples tothe 96-well plate for ease in transferring. Lysates should becentrifuged at 4,600×g for 5 minutes at 4° C. immediately prior to use.Carefully pipette the supernatant. Avoid freeze-thaw cycles. Lysatesshould be diluted at least 10-fold prior to loading onto the assay.

Mouse VEGF Assay Procedure Assay Setup

Perform the Reagent Preparation per instructions included in the kit andbulk reagent package inserts. Prepare the standard curve and the samplesas described above.

Target Capture

After micro particles (MP) have been re-suspended, add 50 μL per well ofVEGF Capture Reagent to 96-well polypropylene plate (PPP). Pipette 10 μLper well of Standards/Samples to 96-well PPP. Pulse spin the plate up to100×g to ensure all of sample is in the MP mixture. Seal plate with atemporary plate seal (AxySeal, PCRSP Plate Sealing Film) or equivalent.Incubate/shake at medium setting for 2 hours at room temperature (RT).Carefully remove temporary plate seal to avoid splashing. Set plate ontomagnet (Dynal MPC®-96S), wait 2 minutes for MP to settle (ensure all MPare amassed as a pellet by magnet), then aspirate the supernatant (MPremain visible). With the MP secured, add 250 μL of Wash Buffer. Wait 2minutes (MP remain amassed) and aspirate buffer.

Detection

Remove plate from the magnet and add 20 μL of VEGF Detection Reagent toeach well. Seal plate with temporary seal. Pulse in centrifuge up to100×g. Remove the plate from the centrifuge and Incubate/shake for 2hours at (RT). Remove plate seal and set plate onto magnet. Wait 2minutes and aspirate the supernatant. Add and then remove 250 μL of WashBuffer 3 times (3×) while MP are magnetized/amassed. Pause for 2 minutesafter each buffer addition. Do not suspend or remove MP from the magnet.Remove plate from the magnet and add 250 μL of Wash Buffer to each well.Shake plate for 10 seconds to re-suspend MP. Transfer contents of eachwell to a new 96-well PPP. Set new 96-well plate onto magnet and wait 2minutes for MP to amass/settle. Remove Wash Buffer. Remove plate frommagnet, add 250 μL of Wash Buffer and shake for 10 sec. Load plate onmagnet, wait 2 minutes, then aspirate buffer. Repeat cycle, magnetizedMP should be visible.

Elution

Remove plate from the magnet and add 20 μL of per well Elution Buffer.Seal plate with temporary seal and pulse in centrifuge up to 100×g.Incubate/shake for 30 minutes at RT. Separately, place a 384-wellfilter-plate over a 384-well PPP assay plate, making a filter-bottomplate. Remove seal from 96-well plate, transfer specimens to the384-well filter-bottom plate. Cover the top of the filter-bottom platewith temporary plate seal and set plates into centrifuge. Spin plates at850×g for 1 minute at RT. Remove filter plate and discard, cover assayplate using the pierceable (permanent) plate seal (Nunc, 235306). Toensure a good seal, use Plate Seal Roller (VWR #60941-118). Loadcompleted assay plate onto Erenna Immunoassay System.

Mouse VEGF Quick Assay Guide

-   -   1. Prepare all reagents, standard curve, and samples as        instructed.    -   2. Add 50 μL of Capture Reagent, followed by 10 μL of        Standards/Samples to each well of 96-well polypropylene plate.    -   3. Pulse spin plate up to 100×g to ensure samples are in the MP        solution.    -   4. Cover and incubate/shake for two hours at RT.    -   5. Remove cover, set plate onto magnet, allow 2 minutes for MP        to settle/amass and remove supernatant.    -   6. With plate on magnet, add 250 μL of Wash Buffer. Wait 2        minutes and remove buffer.    -   7. Remove from magnet and add 20 μL of VEGF Detection Reagent        per well. Pulse centrifuge at 1000 RPM.    -   8. Cover and incubate/shake for 2 hours at RT.    -   9. Set plate onto magnet and wait 2 minutes for MP to amass.        Remove supernatant.    -   10. Add and then remove 250 μL of Wash Buffer 3× with MP        magnetically amassed near the magnet. Wait 2 minutes before        aspirating the buffer between each cycle.    -   11. Remove from magnet, add 250 μL of Wash Buffer and shake        plate for 10 seconds to re-suspend MP. Transfer entire contents        to new 96-well plate.    -   12. Set plate onto magnet, wait 2 minutes. Remove supernatant.    -   13. Remove from magnet, add 250 μL of Wash Buffer and shake        plate for 10 seconds.    -   14. Repeat steps 11, 12, and 13 respectively.    -   15. Remove from magnet and add 20 μL of Elution Buffer to each        well. Pulse centrifuge at 100×g.    -   16. Cover and incubate/shake at RT for 30 minutes.    -   17. Set a filter plate over 384-well plate (assay plate).    -   18. Transfer contents of 96-well plate to 384-well filter        plate/assay plate combo.    -   19. Cover filter plate combo, centrifuge for 1 minute at 850×g.    -   20. Remove top filter plate and discard. Cover 384-well plate        with pierceable plate seal cover.    -   21. Load the plate onto the Erenna System.        This assay may be used to test various types of plasma and        cellular lysates.

Performance Characteristics Typical Standard Curve

The Standard Curve shown in Table 30 is provided for informationalpurposes. A standard curve should be generated for each set of samplesassayed.

TABLE 30 Standard Curve Expected mVEGF [pg/ml] DE mean std dev cv EPmean std dev cv TP mean std dev cv 0.0  168  28 17%  15207  2114 14% 6163670  87902  1% 3.9  177  27 15%  15807  1406  9%  6239587  98719 2% 7.8  242  26 11%  21854  2342 11%  6360689  80386  1% 15.6  302  11 4%  28145  1949  7%  6429962  44791  1% 31.3  418  42 10%  38805  381810%  6370440  101262  2% 62.5  652  6  1%  62375  2971  5%  6533290 50260  1% 125.0  1112 127 11%  118599 15256 13%  7141792  531505  7%250.0  2104 123  6%  225687 13232  6%  7071139  80642  1% 500.0  3871865 22%  491548 94923 19%  8753982 1946419 22% 1000.0  6693 399  6%1078600 94079  9%  9319958  293189  3% 2000.0  9292 298  3% 214204711297  1% 12032097  166349  1% 4000.0 10193  58  1% 3719950 81130  2%18770298  660699  4% KEY: Detected Events (DE), Event Photons (EP),Total Photons (TP)

Example 18 Highly Sensitive Detection of VEGF

The sensitivity of the system for different concentrations of VEGF inplasma is presented in Table 31. The data is presented graphically inFIG. 25A.

TABLE 31 VEGF-A Curve Fit Data Expected Measured hVEGF hVEGF Standard[pg/ml] [pg/ml] deviation CV Recovery 0.00 ND — — — 0.06 0.08 0.03 41%127% 0.12 0.12 0.02 14% 104% 0.24 0.26 0.03 10% 107% 0.48 0.52 0.04  8%108% 0.98 0.96 0.19 20%  97% 1.95 1.86 0.09  5%  96% 3.90 3.96 0.15%  4%101% 8 9 1.27 15% 111% 16 17 2.09 12% 109% 31 31 2.96 10%  99% 63 621.50  2%  99% 125 123 3.79  3%  98% 250 227 11.61  5%  91% 500 500 18.06 4% 100% 1000 1175 191.67 16% 118%

At the low end of the VEGF-A standard curve the concentration of VEGF-Adetected is shown in Table 32.

TABLE 32 Low-end VEGF-A Standard Curve Data Expected hVEGF Mean Standard[pg/ml] DE deviation CV N 0.00 99 11.4 11%  3 0.06 161 29.3 18%  3 0.12207 17.3 8% 3 0.24 335 26.6 8% 3 0.48 595 43.5 7% 3 0.98 1006 152.6 15% 3 1.95 1771 52.9 3% 3 3.90 3167 101.7 3% 3 7.80 5311 476.3 9% 3 15.607597 362.1 5% 3

This data corresponds to the graph shown in FIG. 25B.

Example 19 Measured Versus Expected Values for VEGF

FIG. 26 shows measured versus expected values for VEGF in threedifferent assay formats. Standard calibration curves for the three humanVEGF assays using different solid phase immunoassay formats were run ona common set of serially diluted calibrators. The hVEGF MP-based assayuses paramagnetic microparticles coated with detection antibody as thesolid phase capture format, and a fluorescently labeled detectionantibody. The hVEGF Plate-based assay uses a uses 384-well plate, wherewells have been coated with detection antibody as the solid phasecapture format, and a fluorescently labeled detection antibody. ThehVEGF HRP-ELISA assay is a commercially available ELISA assay from R&DSystems (LoD=31.2 pg/mL) consisting of a 96-well solid phase captureformat, and uses an enzymatically conjugated detection antibody.

Example 20 Detection of VEGF in Plasma and Cell Lysate Small VolumeSamples

The levels of human VEGF detected in 10 μl samples from healthy andbreast cancer patients were compared. The limit of detection (LOD) usingthe method of the present invention (Errena; LOD=3.5 pg/ml) versus astandard ELISA format (LOD=31.2 pg/ml) is shown. Human plasma (FIG. 27A)and tissue (FIG. 27B) samples were tested with the Erenna hVEGF-Aimmunoassay. (FIG. 27A) Circulating concentration of hVEGF-A wasdetermined in plasma samples from healthy blood donors (n=24) andsubjects with breast cancer (n=15). The median and interquartile rangeof plasma VEGF levels were calculated, and compared between healthyblood donors and subjects with breast cancer. (FIG. 27B) Comparison ofmedian and interquartile range of matched malignant and non-malignanttissue biopsy samples from subjects with breast cancer (n=10). Tissuesamples were designated post-surgically as either normal or malignant,and results are shown in pg of VEGF protein per mg of total protein persample. Quantification of the plasma samples with the present inventionincluded all samples tested from healthy and cancerous subjects, whilequantification using the standard ELISA assay showed poor quantificationof healthy samples. Similar to the case in plasma, quantification oftissue samples with the present invention included all samples testedfrom healthy and cancerous subjects, while quantification using thestandard ELISA assay showed poor quantification of healthy samples.

Example 21 Combined Analog and Digital Measurements of VEGF

FIG. 28 shows the correlation of readout methods for the presentinvention. A standard curve was generated for the hVEGF analyte andmeasured with the Erenna system. Results are shown for each of threedifferent read-out methods: (a) total photons (TP), which is analogousto standard ELISA plate reader technology; (b) detected events (DE),which counts single molecules passing through the interrogation zone asdiscreet events; and (c) using a processing algorithm which combinestotal photons and detected events. (FIG. 28A) and (FIG. 28B) LoD wascalculated using the results of each method (DE and TP) using twostandard deviations of the mean divided by slope. Data in FIG. 28A andFIG. 28B were analyzed using four-parameter curves. Data in FIG. 28C wasanalyzed using linear regression, resulting equations and correlationstatistics are shown.

Example 22 Aβ-40 and Aβ-42 (Amyloid Beta Proteins 40 and 42) Assay

The present invention provides an assay for Aβ-40 and Aβ-42. Thespecification of the system for Aβ-40 and Aβ-42 in a sample is presentedin Table 33.

TABLE 33 Specifications of Singulex Aβ-40 and Aβ-42 assays AttributeAβ-40 Aβ-42 LoD 0.2 pg/ml 0.1 pg/ml LLoQ 0.8 pg/ml 0.5 pg/ml Range0.2-100 pg/ 0.1-250 pg/ml Levels in human 8.1 pg/ml 30.7 pg/ml plasma:average (4.9-11.6 pg/ml) (18.5-351 pg/ml) (range)

The events detected by the system in relation to the analyteconcentrations of Aβ-40 and Aβ-42 are shown in FIG. 129A. FIG. 29B showsthe specificity and linearity of the Aβ-42 assay.

Example 23 Interleukin 1, Alpha (IL-1α) Assay

Sensitivity of an assay provided by the present invention in detectingIL-1α is shown in Table 34. The LoD is typically around 0.1 pg/ml orless. FIG. 30A illustrates a graph corresponding to the data presentedin Table 34.

TABLE 34 IL-1α Curve Fit Data Expected Measured IL-1α IL-1α Standard[pg/ml] [pg/ml] deviation CV Recovery 2000 2019 104 5% 101% 1000 976 546%  98% 500 516 18 4% 103% 250 256 17 7% 102% 125 120 2 2%  96% 63 63 57% 100% 31 31 0.5 1% 100% 16 17 3.42 21%  106% 7.8 8.4 0.40 5% 107% 3.93.9 0.19 5% 100% 1.95 1.94 0.06 3%  99% 0.98 0.98 0.03 3%  98% 0.49 0.50.08 16%  100% 0.24 0.27 0.02 9% 103%

The low end of the IL-1α curve is described in Table 35 and isgraphically represented in FIG. 30B.

TABLE 35 Low-end IL-1α Standard Curve Data IL-1α Detected Standard[pg/ml] Events deviation CV 0.98 1123 22 2% 0.49 832 46 6% 0.24 703 122% 0.12 628 9 1% 0.00 572 28 5%

Example 24 Interleukin 1, Beta (IL-1β) Assay

Sensitivity of one embodiment for different concentrations of IL-1β areshown in Table 36 below. The LoD is typically 0.02 pg/ml or less. Theexpected concentration versus the measured or calculated concentrationof IL-1β is shown graphically in FIG. 31A.

TABLE 36 IL-1β Curve Fit Data Expected Measured IL-1β IL-1β Standard[pg/ml] [pg/ml] deviation CV Recovery 2000 2019 104 5% 101% 1000 976 546%  98% 500 516 18 4% 103% 250 256 17 7% 102% 125 120 2 2%  96% 63 63 57% 100% 31 31 0.5 1% 100% 16 17 3.42 21%  106% 7.8 8.4 0.40 5% 107% 3.93.9 0.19 5% 100% 1.95 1.94 0.06 3%  99% 0.98 0.98 0.03 3%  98% 0.49 0.50.08 16%  100% 0.24 0.27 0.02 9% 103%

The low-end IL-1β standard curve data is presented in Table 37 below.These values are presented graphically in FIG. 31B.

TABLE 37 Low-end IL-1β standard curve data IL-1β Detected Standard[pg/ml] Events deviation CV 3.13 3282 19 0% 1.56 1968 93 1% 0.78 1300 565% 0.39 936 45 4% 0.20 745 36 5% 0.10 691 18 5% 0.05 631 48 3% 0.02 61619 8% 0.01 583 14 3% 0.00 590 45 2%

Example 25 Interleukin 4 (IL-4) Assay

The sensitivity of an IL-4 assay provided by the present invention ispresented in Table 38. The expected IL-4 concentration levels versus thecalculated or measured IL-4 levels are shown in FIG. 32A.

TABLE 38 IL-4 Curve Fit Data Expected Measured IL-4 IL-4 Standard[pg/ml] [pg/ml] deviation CV Recovery 2000 2063 71 3% 103% 1000 1023 505% 102% 500 482 18 4%  96% 250 252 45 18%  101% 125 147 5 3% 117% 63 730 1% 117% 31 29 3 11%   94% 16 13 2 14%   84% 7.8 6.8 1.3 19%   87% 3.93.6 0.1 3%  92% 1.95 1.83 0.10 6%  94% 0.98 1.01 0.11 11%  103% 0.490.58 0.20 3% 118% 0.24 0.36 0.10 28%  146%

The IL-4 assay quantified as little as 0.04 pg/ml of plasma IL-4 with aCV<20%. In some embodiments, the LoD is 0.04 pg/ml. Table 39 lists theconcentrations of IL-4 detected on the low end IL-4 standard curve data.FIG. 32B corresponds to the data presented in Table 39.

TABLE 39 Low-end IL-4 Standard Curve Data IL-4 Detected Standard [pg/ml]Events deviation CV 3.91 1761 44 3% 1.95 1042 50 5% 0.98 674 46 7% 0.49488 7 1% 0.24 392 41 11%  0.12 300 35 12%  0.00 245 8 3%

Example 26 Interleukin 6 (IL-6) Assay

The sensitivity and accuracy of one embodiment of an IL-6 assay providedby the present invention is illustrated in Table 40. The expected IL-6concentration versus the concentration calculated or measured by theassay is depicted graphically in FIG. 33A.

TABLE 40 IL-6 Curve Fit Data Expected Measured IL-6 IL-6 Standard[pg/ml] [pg/ml] deviation CV Recovery 100 119 32.76 28% 119% 50 49 6.9914%  98% 25 22 2.39 11%  90% 12.5 12.8 0.57  4% 102% 6.3 6.9 1.17 17%111% 3.1 3.2 0.21  7% 102% 1.56 1.47 0.03  2%  94% 0.78 0.73 0.04  6% 94% 0.39 0.39 0.02  5% 100% 0.20 0.21 0.02 12% 107% 0.10 0.10 0.02 18%100% 0.05 0.06 0.01 24% 114%

The low-end IL-6 standard curve data is depicted in Table 41 and ispresented graphically in FIG. 33B.

TABLE 41 Low-end IL-6 Standard Curve Data IL-6 Detected Standard [pg/ml]Events deviation CV 1.56 3067 47 2% 0.78 1728 97 6% 0.39 1002 38 4% 0.20589 58 10%  0.10 338 41 12%  0.05 247 30 12%  0.02 168 18 10%  0.01 1376 4% 0 127 8 6%

The IL-6 assay quantifies as little as 0.01 pg/ml of plasma IL-6 at a CVof <20%. The LoD is 0.01 pg/ml or less. This enables the accuratequantification of IL-6 in human plasma, obtained from healthy subjects,with ranges from 0.36-1.17 pg/ml or less.

Example 27 Biomarker Assays

The limits of detection (LODs) of various markers disclosed herein wereassayed according to the present invention. The results of the assaysare presented in Tables 42 and 43. Applications for various markers areindicated in Tables 42, 43 and 44.

TABLE 42 Limits of Detection for Various Biomarkers Biomarker ClassIndications LoD cTnI Cardiac Necrosis 0.01 proBNP Cardiac MyocardialDisfunction 0.03 IL-1-alpha Inflammation 0.07 IL-1-beta InflammationUnstable angina (UAP) 0.01 IL-6 Inflammation Plaques, Heart failure(HF), 0.01 Coronary artery disease (CAD), Myocardial infarction (MI)IL-8 Inflammation UAP 0.36 IL-10 Inflammation Anti-inflammatory 0.46TNF-alpha Inflammation UAP, CAD, HF, Congestive 0.01 heart failure(CHF), MI IFN-gamma Inflammation Rheumatic heart disease (RHD), 0.14auto-immune VEGF Cancer Angiogenesis 0.10 Insulin Metabolic MetabolicSyndrome 12 GLP-1 (T&A) Inflammation Metabolic Syndrome 0.01

TABLE 43 Limits of Detection for Various Cytokines Biomarker IndicationsLoD IL-1-alpha Inflammation 0.07 IL-1-beta UAP 0.01 IL-6 Plaques, HF,CAD, MI 0.01 IL-8 UAP 0.36 IL-10 Anti-inflammatory 0.46 IL-17 IL-21IFN-gamma RA, Systemic lupus erythematosus (SLE), RHD, 0.14auto-immunity Mip1-alpha RANTES TNF-alpha Cancer, Alzheimer's disease(AD), UAP, CAD, HF, 0.01 CHF, MI VEGF Cancer, Angiogenesis,Artherosclreosis, Diabetes 0.10

TABLE 44 Exemplary Marker Indications Assay Neurologic MetabolicOncology Inflammatory IL-1 a X IL-1b X IL-4 X X IL-6 X X X IL-8 X XIL-17 X X X IFN-g X Oxytocin X cAMP X X X X VEGF X TNF-a X X PSA total XPSA free X Ab-40 X Ab-42 X Insulin X GLP-1 X Troponin-1 X X X X TGFb-1 XX X X

Example 28 Biomarker Panels for Cardiovascular Disease Detection

Materials & Methods: EDTA-plasma were collected from subjects with CHF(N=32, 40% female, 76±11 yrs, 56% NYHA I/II, 44% NYHA III) and from anage and sex matched control cohort of apparently healthy subjects (N=32,40% female, mean age 75±10 yrs). Collected panels of EDTA plasma werepurchased from ProMedDx.

Samples were tested for a broad panel of protein biomarkers immunoassaysdeveloped for the Erenna® immunoassay system, which is based upon singlemolecule counting. Assays in the initial multi-panel included: thecardiac pathology markers cTnI and pro-BNP; the vascular inflammationmarker VEGFa; and the inflammatory cytokines IL-1-alpha, IL-6, IL-8,IL-10, IL-17a, IL-17f, TNF-alpha and IFN-gamma. The SOP for a standardErenna immunoassays is described below.

Samples were read in the Erenna instrument, detected events, eventphotons and total photons values were determined, and analyteconcentrations were calculated in pg/mL using SMD Curve Fit, v 2.0 forall samples tested. Hierarchical clustering methods were used to developa training set for inclusion/exclusion, and the resulting panel wasevaluated for diagnostic specificity. Data were arranged into a datamatrix and hierarchical clustering was performed using Cluster v 3.0.Data were log-transformed, centered against the mean, and normalized.Hierarchical clustering of both the protein set and the EDTA plasmasamples was performed using average distance. The initial data set wasused as a training set, and clustering was repeated on successive datasets until diagnostic specificity was optimized.

The resulting panel was further tested for individual significance in acohort comparison between CHF and control subjects using a student'sT-test. Markers with significant differences between the cohorts wereidentified for the final panel. Cut-points determined for eachindividual marker using the mean+3 standard deviations (99% CI) of thecontrol subject values. The number of individuals in the CHF panel aboveand below the cut-points were determined for each individual marker andused to calculate the diagnostic sensitivity of that marker, as well asthe cumulative diagnostic sensitivity of the panel.

The standard operating procedure for the Erenna Immunoassay System is asfollows:

-   -   Construct standard curve dilution series using urea assay buffer        and the appropriate range for the experimental purpose, using        standard analyte    -   Dilute microparticles (MP) in urea assay buffer to optimized        final concentration    -   Combine 100 ul of diluted MP plus 100 ul of standard or sample        in a 96 well polypropylene plate    -   Incubate 2 hrs with shaking    -   Wash to remove unbound proteins    -   Add 20 ul of filtered detection Ab diluted in TBS/BSA based        assay buffer with goat/mouse/rabbit IgG+NaF    -   Incubate 1 hr with shaking    -   Wash to remove unbound detection Ab    -   Add 20 ul elution reagent/well and incubate with shaking for 10        min    -   Transfer to 384 well filter plate    -   Spin filter into 384 well polypropylene plate containing 4 ul of        1M Tris    -   Read in Erenna System

Results: Hierarchical clustering of the training set yielded apreliminary multi-marker panel with 91% diagnostic specificity for CHFcompared to age and sex matched control subjects (FIG. 34). A subset ofmarkers were further identified that showed significant differencesbetween the two cohorts: cTnI (p=0.0025), BNP (p<0.0001), IL-6(p<0.0001), TNFα (p<0.0001) and IL-17a (p=0.0166). These markers wereused for the final panel, cut-points and diagnostic specificities weredetermined (TABLE 35). Based on established cut-points, diagnosticsensitivity of the individual markers was low compared to the cumulativesensitivity of the panel. Inclusion of all 5 protein biomarkers in thepanel yielded a combined diagnostic sensitivity of 94%. In addition,multiple biomarkers were elevated for 75% (24/32) subjects in the CHFpanel (FIG. 35) suggesting that synergistic biomarker elevation is acontributing factor.

TABLE 35 Individual and cumulative diagnostic sensitivity of the CVDbiomarker panel Marker cTnI BNP IL-6 TNFα IL-17a Cutpoint (pg/ml) 8.0241 4.6 6.68 2.65 # below 17 13 23 15 28 # above 15 19 9 17 4Sensitivity (alone) 47% 59% 28% 53% 13% (cumulative) — 66% 78% 91% 94%

What is claimed is:
 1. A method for detecting or monitoring acardiovascular condition in a subject, comprising detecting two or morebiomarkers in a sample from the subject, wherein the biomarkers arecardiac pathology biomarkers selected from the group consisting ofTroponin-I (cTnI), B-type Natriuretic Peptide (BNP), proBNP andNT-proBNP, and vascular inflammation biomarkers selected from the groupconsisting of Interleukin 6 (IL-6), Tumor Necrosis Factor alpha (TNF-α),or Interleukin 17a (IL-17a), and wherein the limit of detection of atleast one biomarker is less than about 20 pg/ml.
 2. The method of claim1, wherein the detection of the at least one biomarker having the limitof detection less than about 20 pg/ml comprises contacting the samplewith a label for the marker and detecting the presence or absence of thelabel, wherein detection of the presence of the label indicates thepresence of the corresponding marker.
 3. The method of claim 2, whereinthe label comprises a fluorescent moiety, and the at least one biomarkerhaving the limit of detection less that about 20 pg/nl is detected in aninterrogation space comprising a label corresponding to a singlemolecule of the biomarker.
 4. The method of claim 2, wherein the labelcomprises a fluorescent moiety, and the detection comprises passing thelabel through a single molecule detector, wherein the single moleculedetector comprises: (a) an electromagnetic radiation source forstimulating the fluorescent moiety; (b) an interrogation space forreceiving electromagnetic radiation emitted from the electromagneticsource; and (c) an electromagnetic radiation detector operably connectedto the interrogation space for determining an electromagneticcharacteristic of the stimulated fluorescent moiety.
 5. The method ofclaim 1, wherein the limit of detection of at least one marker rangesfrom about 10 pg/ml to about 0.01 pg/ml.
 6. The method of claim 1,wherein the condition comprises congestive heart failure and the two ormore biomarkers comprise cTnI, BNP, IL-6, TNF-α, and IL-17a.
 7. A methodto detect a cardiovascular condition in a subject, comprising measuringa physiological biomarker and detecting one or more biomarkers in ablood sample from the subject, wherein the biomarkers are selected fromthe group consisting Troponin-I (cTnI), B-type Natriuretic Peptide(BNP), proBNP, NT-proBNP, Interleukin 6 (IL-6), Tumor Necrosis Factoralpha (TNF-α), or Interleukin 17a (IL-17a), and wherein the limit ofdetection of at least one biomarker is less than about 20 pg/ml.
 8. Themethod of claim 7, wherein the physiological biomarker is a stress test.9. The method of claim 8, wherein the one or more biomarkers comprisecTnI.
 10. The method of claim 7, wherein the physiological biomarker isa sleep test.
 11. The method of claim 10, wherein the one or morebiomarkers comprise one or more cytokines.
 12. The method of claim 11wherein the one or more cytokines comprises TNF-α, IL-1, IL-6, IL-17aand/or hsCRP.
 13. The method of claim 7, wherein the physiologicalbiomarker is Carotid intima-media thickness (CIMT).
 14. The method ofclaim 13, wherein the one or more biomarkers comprise one or moremarkers of vascular inflammation.
 15. The method of claim 14, the one ormore markers of vascular inflammation comprise hsCRP, IL-6, TNF-a and/orIL-1.
 16. A method for detecting congestive heart failure in a subject,comprising detecting whether one or more members of a panel ofbiomarkers comprising two or more biomarkers are elevated compared tothe level of the one or more biomarkers in a normal population, whereinthe biomarkers are selected from the group consisting of Troponin-I(cTnI), B-type Natriuretic Peptide (BNP), proBNP, NT-proBNP, Interleukin6 (IL-6), Tumor Necrosis Factor alpha (TNF-α), or Interleukin 17a(IL-17a), and wherein the limit of detection of at least one member ofthe panel is less than about 20 pg/ml.
 17. The method of claim 16,wherein the at least one member of the panel having a detection limitless than about 20 pg/ml is cTnI.
 18. The method of claim 16, whereinthe detection of the at least one biomarker having the limit ofdetection less than about 20 pg/ml comprises contacting the sample witha label for the marker and detecting the presence or absence of thelabel, wherein detection of the presence of the label indicates thepresence of the corresponding marker.
 19. The method of claim 16,wherein the label comprises a fluorescent moiety, and the at lease onebiomarker having the limit of detection less that about 20 pg/nl isdetected in an interrogation space comprising a label corresponding to asingle molecule of the biomarker.
 20. The method of claim 16, whereinthe label comprises a fluorescent moiety, and the detection comprisespassing the label through a single molecule detector, wherein the singlemolecule detector comprises: (a) an electromagnetic radiation source forstimulating the fluorescent moiety; (b) an interrogation space forreceiving electromagnetic radiation emitted from the electromagneticsource; and (c) an electromagnetic radiation detector operably connectedto the interrogation space for determining an electromagneticcharacteristic of the stimulated fluorescent moiety.
 21. The method ofclaim 20, wherein the limit of detection of at least one marker rangesfrom about 10 pg/ml to about 0.01 pg/ml.